Methods for blocking binding of CD28 receptor to B7

ABSTRACT

The invention identifies the B7 antigen as a ligand that is reactive with the CD28 receptor on T cells. The invention further provides methods for using antibodies to B7, or fragments thereof, to regulate CD28 positive T cell response and immune responses mediated by T cells.

This is a divisional of U.S. Ser. No. 08/219,200, filed Mar. 29, 1994,now U.S. Pat. No. 6,641,809, which is a FWC Ser. No. 07/722,101, filedJun. 27, 1991, now abandoned, which was a continuation-in-part of Ser.No. 07/547,980 filed Jul. 2, 1990, now abandoned, which was acontinuation-in-part of Ser. No. 07/498,949 filed Mar. 26, 1990, nowabandonded.

FIELD OF THE INVENTION

The present invention relates to the identification of an interactionbetween the CD28 receptor and its ligand, the B7 antigen, and to amethod for regulating cellular interactions using the antigen, fragmentsand derivatives thereof.

BACKGROUND OF THE INVENTION

The generation of a T lymphocyte (“T cell”) immune response is a complexprocess involving cell—cell interactions (Springer et al., A. Rev.Immunol. 5:223-252 (1987)), particularly between T and B cells, andproduction of soluble immune mediators (cytokines or lymphokines)(Dinarello and Mier, New Engl. Jour. Med. 317:940-945 (1987)). Thisresponse is regulated by several T-cell surface receptors, including theT-cell receptor complex (Weiss et al., Ann. Rev. Immunol. 4:593-619(1986)) and other “accessory” surface molecules (Springer et al., (1987)supra). Many of these accessory molecules are naturally occurring cellsurface differentiation (CD) antigens defined by the reactivity ofmonoclonal antibodies on the surface of cells (McMichael, Ed., LeukocyteTyping III, Oxford Univ. Press, Oxford, N.Y. (1987)).

One such accessory molecule is the CD28 antigen, a homodimericglycoprotein of the immunoglobulin superfamily (Aruffo and Seed, Proc.Natl. Acad. Sci. 84:8573-8577 (1987)) found on most mature human T cells(Damle et al., J. Immunol. 131:2296-2300 (1983)). Current evidencesuggests that this molecule functions in an alternative T cellactivation pathway distinct from that initiated by the T-cell receptorcomplex (June et al., Mol. Cell. Biol. 7:4472-4481 (1987)). Monoclonalantibodies (mAbs) reactive with CD28 antigen can augment T cellresponses initiated by various polyclonal stimuli (reviewed by June etal., supra). These stimulatory effects may result from mAb-inducedcytokine production (Thompson et al., Proc. Natl. Acad. Sci.86:1333-1337 (1989); Lindsten et al., Science 244:339-343 (1989)) as aconsequence of increased mRNA stabilization (Lindstein et al., (1989),supra). Anti-CD28 mAbs can also have inhibitory effects, i.e., they canblock autologous mixed lymphocyte reactions (Damle et al., Proc. Natl.Acad. Sci. 78:5096-6001 (1981)) and activation of antigen-specific Tcell clones (Lesslauer et al., Eur. J. Immunol. 16:1289-1296 (1986)).

The in vivo function of CD28 antigen is not known, although itsstructure (Aruffo and Seed, (1987), supra) suggests that like othermembers of the immunoglobulin superfamily (Williams and Barclay, Ann.Rev. Immunol. 6:381-405 (1988), it might function as a receptor. CD28antigen could conceivably function as a cytokine receptor, although thisseems unlikely since it shares no homology with other lymphokine orcytokine receptors (Aruffo and Seed, (1987) supra).

Alternatively, CD28 might be a receptor which mediates cell—cell contact(“intracellular adhesion”). Antigen-independent intercellularinteractions involving lymphocyte accessory molecules are essential foran immune response (Springer et al., (1987), supra). For example,binding of the T cell-associated protein, CD2, to its ligand LFA-3, awidely expressed glycoprotein (reviewed in Shaw and Shimuzu, CurrentOpinion in Immunology, Eds. Kindt and Long, 1:92-97 (1988)), isimportant for optimizing antigen-specific T cell activation (Moingeon etal., Nature 339:314 (1988)). Another important adhesion system involvesbinding of the LFA-1 glycoprotein found on lymphocytes, macrophages, andgranulocytes (Springer et al., (1987), supra; Shaw and Shimuzu (1988),supra) to its ligands ICAM-1 (Makgoba et al., Nature 331:86-88 (1988))and ICAM-2 (Staunton et al., Nature 339:61-64 (1989)). The T cellaccessory molecules CD8 and CD4 strengthen T cell adhesion byinteraction with MHC class I (Norment et al., Nature 336:79-81 (1988))and class II (Doyle and Strominger, Nature 330:256-259 (1987))molecules, respectively. “Homing receptors” are important for control oflymphocyte migration (Stoolman, Cell 56:907-910 (1989)). The VLAglycoproteins are integrins which appear to mediate lymphocyte functionsrequiring adhesion to extracellular matrix components (Hemler,Immunology Today 9:109-113 (1988)). The CD2/LFA-3, LFA-1/ICA-1 andICAM-2, and VLA adhesion systems are distributed on a wide variety ofcell types (Springer et al., (1987), supra; Shaw and Shimuzu, (1988,)supra and Hemler, (1988), supra).

Intercellular adhesion interactions mediated by integrins are stronginteractions that may mask other intercellular adhesion interactions.For example, interactions mediated by integrins require divalent cations(Kishimoto et al., Adv. Immunol. 46:149-182 (1989). These interactionsmay mask other intercellular adhesion interactions that are divalentcation independent. Therefore, it would be useful to develop assays thatpermit identification of non-integrin mediated ligand/receptorinteractions.

T cell interactions with other cells such as B cells are essential tothe immune response. Levels of many cohesive molecules found on T cellsand B cells increase during an immune response (Springer et al., (1987),supra; Shaw and Shimuzu, (1988), supra; Hemler (1988), supra). Increasedlevels of these molecules may help explain why activated B cells aremore effective at stimulating antigen-specific T cell proliferation thanare resting B cells (Kaiuchi et al., J. Immunol. 131:109-114 (1983);Kreiger et al., J. Immunol 135:2937-2945 (1985); McKenzie, J. Immunol.2907-2911 (1988); and Hawrylowicz and Unanue, J. Immunol. 141:4083-4088(1988)). The fact that anti-CD28 mAbs inhibit mixed lymphocyte reactions(MLR) may suggest that the CD28 antigen is also an adhesion molecule.

Optimal activation of B lymphocytes and their subsequent differentiationinto iminunoglobulin secreting cells is dependent on the helper effectsof major histocompatibility complex (MHC) class II antigen (Ag)-reactiveCD4 positive T helper (CD4⁺ T_(h)) cells and is mediated via both direct(cognate) T_(h)-B cell intercellular contact-mediated interactions andthe elaboration of antigen-nonspecific cytokines (non-cognateactivation; see, e.g. Noel and Snow, Immunol. Today 11:361 (1990)).Although T_(h)-derived cytokines can stimulate B cells (Moller, Immunol.Rev. 99:1 (1987)), their synthesis and directional exocytosis isinitiated and sustained via cognate interactions between antigen-primedT_(h) cells and antigen-presenting B cells (Moller, supra). Thesuccessful outcome of T_(h)-B interactions requires participation oftransmembrane receptor-ligand pairs of co-stimulatory accessory/adhesionmolecules on the surface of T_(h) and B cells which include CD2 (LFA-2);CD58 (LFA-3), CD4:MHC class II, CD11a/CD18 (LFA-1):CD54 (1CAM-1).

During cognate T_(h):B interaction, although both T_(h) and B cellscross-stimulate each other, their functional differentiation iscritically dependent on the provision by T_(h) cells of growth anddifferentiation-inducing cytokines such as IL-2, IL-4 and IL-6 (Noel,supra, Kupfer et al., supra, Brian, supra and Moller, supra). Studies byPoo et al. (Nature 332:378 (1988)) on cloned T_(h):B interactionindicate that interaction of the T cell receptor complex (TcR) withnominal Ag-MHC class II on B cells results in focused release of T_(h)cell-derived cytokines in the area of T_(h) and B cell contact(vectorially oriented exocytosis). This may ensure the activation ofonly B cells presenting antigen to T_(h) cells, and also avoidsactivation of bystander B cells.

It was proposed many years ago that B lymphocyte activation requires twosignals (Bretscher and Cohn, Science 169:1042-1049 (1970)) and now it isbelieved that all lymphocytes require two signals for their optimalactivation, an antigen specific or clonal signal, as well as a second,antigen non-specific signal (Janeway, supra). The signals required for aT helper cell (T_(h)) antigenic response are provided byantigen-presenting cells (APC). The first signal is initiated byinteraction of the T cell receptor complex (Weiss, J. Clin. Invest.86:1015 (1990)) with antigen presented in the context of class II majorhistocompatibility complex (MHC) molecules on the APC (Allen, Immunol.Today 8:270 (1987)). This antigen-specific signal is not sufficient togenerate a full response, and in the absence of a second signal mayactually lead to clonal inactivation or anergy (Schwartz, Science248:1349 (1990)). The requirement for a second “costimulatory” signalprovided by the MHC has been demonstrated in a number of experimentalsystems (Schwartz, supra; Weaver and Unanue, Immunol. Today 11:49(1990)). The molecular nature of these second signal(s) is notcompletely understood, although it is clear in some cases that bothsoluble molecules such as interleukin (IL)-1 (Weaver and Unanue, supra)and membrane receptors involved in intercellular adhesion (Springer,Nature 346:425 (1990)) can provide costimulatory signals.

Freeman et al. (J. Immunol. 143(8):2714-2722 (1989)) isolated andsequenced a cDNAclone encoding a B cell activation antigen recognized bymAb B7 (Freeman et al., J. Immunol. 138:3260 (1987)). COS cellstransfected with this cDNA have been shown to stain by both labeled mAbB7 and mAb BB-1 (Clark et al., Human Immunol. 16:100-113 (1986); Yokochiet al., J. Immunol. 128:823 (1981)); Freeman et al., (1989) supra; andFreedman et al., (1987), supra)). Expression of the B cell activationantigen has been detected on cells of other lineages. For example,studies by Freeman et al. (1989) have shown that monocytes express lowlevels of mRNA for B7.

Expression of soluble derivatives of cell-surface glycoproteins in theimmunoglobulin gene superfamily has been achieved for CD4, the receptorfor HIV-1, using hybrid fusion molecules consisting of DNA sequencesencoding portions of the extracellular domain of CD4 receptor fused toantibody domains (human immunoglobulin C gamma 1), as described by Caponet al., Nature 337:525-531 (1989).

While the CD28 antigen has functional and structural characteristics ofa receptor, until now, a natural ligand for this molecule has not beenidentified. It would be useful to identify ligands that bind with theCD28 antigen and other receptors and to use such ligand(s) to regulatecellular responses, such as T cell and B cell interactions, for use intreating pathological conditions.

SUMMARY OF THE INVENTION

Accordingly, the present invention identifies the B7 antigen as a ligandrecognized by the CD28 receptor. The B7 antigen, or its fragments orderivatives are reacted with CD28 positive T cells to regulate T cellinteractions with other cells. Alternatively, CD28 receptor, itsfragments or derivatives are reacted with B7 antigen to regulateinteractions of B7 positive cells with T cells. In addition, antibodiesor other molecules reactive with the B7 antigen or CD28 receptor may beused to inhibit interaction of cells associated with these molecules,thereby regulating T cell responses.

A preferred embodiment of the invention provides a method for regulatingCD28 specific T cell interactions by reacting CD28 positive T cells withB7 antigen, or its fragments or derivatives, so as to block thefunctional interaction of T cells with other cells. The method forreacting a ligand for CD28 with T cells may additionally include the useof anti-CD monoclonal antibodies such as anti-CD2 and/or anti-CD3monoclonal antibody.

In an alternative embodiment, the invention provides a method forregulating immune responses by contacting CD28 positive T cells withfragments containing at least a portion of the DNA sequence encoding theamino acid sequence corresponding to the extracellular domain of B7antigen. In addition, derivatives of B7 antigen may be used to regulateimmune responses, wherein the derivatives are fusion protein constructsincluding at least a portion of the extracellular domain of B7 antigenand another protein, such as human immunoglobulin C gamma 1, that altersthe solubility, binding affinity and/or valency of B7 antigen. Forexample, in a preferred embodiment, DNA encoding amino acid residuesfrom about position 1 to about position 215 of the sequencecorresponding to the extracellular domain of B7 antigen is joined to DNAencoding amino acid residues of the sequences corresponding to thehinge, CH2 and CH3 regions of human Ig Cγ1 to form a DNA fusion productwhich encodes B7Ig fusion protein.

In another preferred embodiment, DNA encoding amino acid residues fromabout position 1 to about position 134 of the sequence corresponding tothe extracellular domain of the CD28 receptor is joined to DNA encodingamino acid residues of the sequences corresponding to the hinge, CH2 andCH3 regions of human Ig Cγ1 to form a CD28Ig fusion protein.

Alternatively, fragments or derivatives of the CD28 receptor may bereacted with B cells to bind the B7 antigen and regulate T cell/B cellinteractions. The methods for regulating T cell interactions may befurther supplemented with the addition of a cytokine.

In another embodiment, the invention provides a method for treatingimmune system diseases mediated by T cell by administering B7 antigen,including B7Ig fusion protein, to react with T cells by binding the CD28receptor.

In yet another embodiment, a method for inhibiting T cell proliferationin graft versus host disease is provided wherein CD28 positive T cellsare reacted with B7 antigen, for example in the form of the B7Ig fusionprotein, to bind to the CD28 receptor, and an immunosuppressant isadministered.

The invention also provides a cell adhesion assay to identify ligandsthat interact with target receptors that mediate intercellular adhesion,particularly adhesion that is divalent cation independent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are bar graphs showing the results of cellular adhesionexperiments using CD28 positive (CD28⁺⁾ and CD28 negative (CD28⁻) CHOcells as described in Example 1, infra.

FIG. 2 are micrographs of the cellular adhesion studies of FIG. 1, asdescribed in Example 1, infra.

FIG. 3 are bar graphs of experiments testing the ability of differenthuman cell lines and normal and activated murine spleen B cells toadhere to CD28⁺ CHO cells, as described in Example 1, infra.

FIG. 4 is a graph of the effects of blocking by mAbs on CD28-mediatedadhesion to human B cells, as described in Example 1, infra.

FIG. 5 is a bar graph of the results of adhesion between COS cellstransfected with B7 antigen and CD28⁺ or CD28⁻ CHO cells, as describedin Example 1, infra.

FIG. 6 is a bar graph demonstrating the effect of anti-CD28 and anti-B7mAbs on T cell proliferation as described in Example 2, infra.

FIG. 7 a is a graph showing the effects of DR7-primed CD4⁺CD45RO⁺ T_(h)cells on differentiation of B cells into IgM secreting SKW B cells, asdescribed in Example 2, infra.

FIG. 7 b is a graph showing the effects of DR7-primed CD4⁺CD45RO⁺ T_(h)cells on differentiation of B cells into IgG secreting CESS B cells, asdescribed in Example 2, infra.

FIG. 8 a is a graph showing the effect of anti-CD28 and anti-B7 mAbs onthe T_(h)-induced production of IgM by B cells as described in Example2, infra.

FIG. 8 b is a graph showing the effect of anti-CD28 and anti-B7 mAbs onthe T_(h)-induced production of IgG by B cells as described in Example2, infra.

FIG. 9 a is a diagrammatic representation of B7Ig protein fusionconstructs as described in Example 3, infra (dark shadedregions=oncostatin M; unshaded regions=B7, stippled regions=human IgCγ1).

FIG. 9 b is a diagrammatic representation of CD28Ig protein fusionconstructs as described in Example 3, infra (dark shadedregions=oncostatin M; unshaded regions=CD28, stippled regions=human IgCγ1).

FIG. 10 is a photograph of a gel obtained from purification of B7Ig andCD28 protein fusion constructs as described in Example 3 infra.

FIG. 11 depicts the results of FACS^(R) analysis of binding of the B7Igand CD28Ig fusion proteins to transfected CHO cells as described inExample 3, infra.

FIG. 12 is a graph illustrating competition binding analysis of¹²⁵I-labeled B7Ig fusion protein to immobilized CD28Ig fusion protein asdescribed in Example 3, infra.

FIG. 13 is a graph showing the results of Scatchard analysis of B7Igfusion protein binding to immobilized CD28Ig fusion protein as describedin Example 3, infra.

FIG. 14 is a graph of FACS^(R) profiles of B7Ig fusion protein bindingto PHA blasts as described in Example 3, infra.

FIG. 15 is an autoradiogram of ¹²⁵I-labeled proteins immunoprecipitatedby B7Ig as described in Example 3, infra.

FIG. 16 is a graph showing the effect of B7Ig binding to CD28 onCD28-mediated adhesion as described in Example 3, infra.

FIG. 17 is a photograph of the results of RNA blot analysis of theeffects of B7 on accumulation of IL-2 mRNA as described in Example 3,infra.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

This invention is directed to the identification of a ligand reactivewith CD28 antigen (hereafter referred to as “CD28 receptor”), and tomethods of using the ligand and its fragments and derivatives, includingfusion proteins. Also disclosed is a cell adhesion assay method todetect ligands for cell surface receptors.

Recently, Freeman et al., (J. Immunol. 143 (8): 2714-2722 (1989))isolated and sequenced a cDNA clone encoding a B cell activation antigenrecognized by monoclonal antibody (mAb) B7 (Freedman et al., J. Immunol.139:3260 (1987)). COS cells transfected with this cDNA were shown tostain by both mAb B7 and mAb BB-1 (Clark et al., Human Immunology16:100-113 (1986), and Yokochi et al., (1981), supra Freeman et al.,(1989) supra; and Freedman et al., (1987), supra). The ligand for CD28was identified by the experiments described herein, as the B7/BB-1antigen isolated by Freeman et al., wherein the predicted amino acidsequence of amino acid 1-216 are:

Gly Leu Ser His Phe Cys Ser Gly Val Ile His Val1               5              10 Thr Lys Glu Val Lys Glu Val Ala ThrLeu Ser Cys        15                    20 Gly His Asn Val Ser Val GluGlu Leu Ala Gln Thr  25                  30                  35 Arg IleTyr Trp Gln Lys Glu Lys Lys Met Val Leu             40                  45 Thr Met Met Ser Gly Asp Met Asn IleTrp Pro Glu      50                  55                  60 Tyr Lys AsnArg Thr Ile Phe Asp Ile Thr Asn Asn                 65                  70 Leu Ser Ile Val Ile Leu Ala LeuArg Pro Ser Asp          75                  80 Glu Gly Thr Tyr Glu CysVal Val Leu Lys Tyr Glu  85                  90                  95 LysAsp Ala Phe Lys Arg Glu His Leu Ala Glu Val            100                 105 Thr Leu Ser Val Lys Ala Asp Phe ProThr Pro Ser     110                 115                 120 Ile Ser AspPhe Glu Ile Pro Thr Ser Asn Ile Arg                125                 130 Arg Ile Ile Cys Ser Thr Ser GlyGly Phe Pro Glu         135                 140 Pro His Leu Ser Trp LeuGlu Asn Gly Glu Glu Leu 145                 150                 155 AsnAla Ile Asn Thr Thr Val Ser Gln Asp Pro Glu            160                 165 Thr Glu Leu Tyr Ala Val Ser Ser LysLeu Asp Phe     170                 175                 180 Asn Met ThrThr Asn His Ser Phe Met Cys Leu Ile                185                 190 Lys Tyr Gly His Leu Arg Val AsnGln Thr Phe Asn         195                 200 Trp Asn Thr Thr Lys GlnGlu His Phe Pro Asp Asn 205                 210                 215

(Freedman et al., and Freeman et al., supra, both of which areincorporated by reference herein).

For convenience, the ligand for CD28, identified as the B7/BB-1 antigen,is referred to herein as the “B7 antigen”.

The term “fragment”as used herein means a portion of the amino acidsequence corresponding to the B7 antigen or CD28 receptor. For example,a fragment of the B7 antigen useful in the method of the presentinvention is a polypeptide containing a portion of the amino acidsequence corresponding to the extracellular portion of the B7 antigen,i.e. the DNA encoding amino acid residues from position 1 to 215 of thesequence corresponding to the B7 antigen described by Freeman et al.supra A fragment of the GD28 antigen that may be used is a polypeptidecontaining amino acid residues from about position 1 to about position134 of the sequence corresponding to the CD28 receptor as described byAruffo and Seed, Proc. Natl. Acad. Sci. (USA) 84:8573-8577 (1987). Theterm “derivative”as used herein includes a fusion protein consisting ofa polypeptide including portions of the amino acid sequencecorresponding to the B7 antigen or CD28 antigen. For example, aderivative of the B7 antigen useful in the method of the presentinvention is a B7Ig fusion protein that comprises a polypeptidecorresponding to the extracellular domain of the B7 antigen and animmunoglobulin constant region that alters the solubility, affinityand/or valency (valency is defined herein as the number of binding sitesavailable per molecule) of the B7 antigen.

The term “derivative” also includes monoclonal antibodies reactive withthe B7 antigen or CD28 receptor, or fragments thereof, and antibodiesreactive with the B7Ig and CD28Ig fusion proteins of the invention.

The B7 antigen and/or its fragments or derivatives for use in thepresent invention may be produced in recombinant form using knownmolecular biology techniques based on the cDNA sequence published byFreeman et al., supra. Specifically, cDNA sequences encoding the aminoacid sequence corresponding to the B7 antigen or fragments orderivatives thereof can be synthesized by the polymerase chain reaction(see U.S. Patent No. 4,683,202) using primers derived from the publishedsequence of the antigen (Freeman et al., supra). These cDNA sequencescan then be assembled into a eukaryotic or prokaryotic expression vectorand the resulting vector can be used to direct the synthesis of theligand for CD28 by appropriate host cells, for example COS or CHO cells.CD28 receptor and/or its fragments or derivatives may also be producedusing recombinant methods. In a preferred embodiment, DNA encoding theamino acid sequence corresponding to the extracellular domain of the B7antigen, containing amino acids from about position I to about position215, is joined to DNA encoding the amino acid sequences corresponding tothe hinge, CR2 and CH3 regions of human Ig Cyl, using PCR, to form aconstruct that is expressed as B7Ig fusion protein. DNA encoding theamino acid sequence corresponding to the B7Ig fusion protein has beendeposited with the American Type Culture Collection (ATCC) in Rockville,Maryland, under the Budapest Treaty on May 31, 1991 and accordedaccession number 68627.

In another embodiment, DNA encoding the amino acid sequencecorresponding to the extracellular domain of the CD28 receptor,containing amino acids from about position 1 to about position 134, isjoined to DNA encoding the amino acid sequences corresponding to thehinge, CR2 and CH3 regions of human Ig Cγ1 using PCR to form a constructexpressed as CD28Ig fusion protein. DNA encoding the amino acid sequencecorresponding to the CD28Ig fusion protein has been deposited in theATCC, in Rockville, Maryland under the Budapest Treaty on May 31, 1991and accorded accession number 68628.

The techniques for assembling and expressing DNA encoding the amino acidsequences corresponding to B7 antigen and soluble B7Ig and CD28Ig fusionproteins, e.g synthesis of oligonucleotides, PCR, transforming cells,constructing vectors, expression systems, and the like arewell-established in the art, and most practitioners are familiar withthe standard resource materials for specific conditions and procedures.Rowever, the following paragraphs are provided for convenience andnotation of modifications where necessary, and may serve as a guideline.

Cloning and Expression of Coding Seuuences for Receptors and FusionProteins cDNA clones containing DNA encoding CD28 and B7 proteins areobtained to provide DNA for assembling CD28 and B7 fusion proteins asdescribed by Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84:8573-8579(1987) (for CD28); and Freeman et al., J. Immunol. 143:2714-2722 (1989)(for B7), incorporated by reference herein. Alternatively, cDNA clonesmay be prepared from RNA obtained from cells expressing B7 antigen andCD28 receptor based on knowledge of the published sequences for theseproteins (Aruffo and Seed, and Freeman, supra) using standardprocedures. The cDNA is amplified using the polymerase chain reaction(“PCR”) technique (see U.S. Patent Nos. 4,683,195 and 4,683,202 toMullis et al. and Mullis & Faloona, Methods Enzymol. 154:335-350 (1987))using synthetic oligonucleotides encoding the sequences corresponding tothe extracellular domain of the CD28 and B7 proteins as primers. PCR isthen used to adapt the fragments for ligation to the DNA encoding aminoacid fragments corresponding to the human immunoglobulin constant γ 1region, i.e. sequences encoding the hinge, CH2 and CH3 regions of Ig Cγlto form B7Ig and CD28Ig fusion constructs and to expression plasmid DNAto form cloning and expression plasmids containing sequencescorresponding to B7 or CD28 fusion proteins.

To produce large quantities of cloned DNA, vectors containing DNAencoding the amino acid sequences corresponding to the fusion constructsof the invention are transformed into suitable host cells, such as thebacterial cell line MC1061/p3 using standard procedures, and coloniesare screened for the appropriate plasmids.

The clones obtained as described above are then transfected intosuitable host cells for expression. Depending on the host cell used,transfection is performed using standard techniques appropriate to suchcells. For example, transfection into mammalian cells is accomplishedusing DEAE-dextran mediated transfection, CaPO₄ co-precipitation,lipofection, electroporation, or protoplast fusion, and other methodsknown in the art including: lysozyme fusion or erythrocyte fusion,scraping, direct uptake, osmotic or sucrose shock, directmicroinjection, indirect microinjection such as via erythrocyte-mediated techniques, and/or by subjecting host cells to electriccurrents. The above list of transfection techniques is not considered tobe exhaustive, as other procedures for introducing genetic informationinto cells will no doubt be developed.

Expression plasmids containing cDNAs encoding sequences corresponding toCD28 and B7 for cloning and expression of CD28Ig and B7Ig fusionproteins include the OMCD28 and OMB7 vectors modified from vectorsdescribed by Aruffo and Seed, Proc. Natl. Acad. Sci. USA (1987), supra,(CD28); and Freeman et al., (1989), supra, (B7), both of which areincorporated by reference herein. Preferred host cells for expression ofCD28Ig and B7Ig proteins include COS and CHO cells.

Expression in eukaryotic host cell cultures derived from multicellularorganisms is preferred (see Tissue Cultures, Academic Press, Cruz andPatterson, Eds. (1973)). These systems have the additional advantage ofthe ability to splice out introns and thus can be used directly toexpress genomic fragments. Useful host cell lines include Chinesehamster ovary (CHO), monkey kidney (COS), VERO and HeLa cells. In thepresent invention, cell lines stably expressing the fusion constructsare preferred.

Expression vectors for such cells ordinarily include promoters andcontrol sequences compatible with mammalian cells such as, for example,CMV promoter (CDM8 vector) and avian sarcoma virus (ASV) (πLN vector).Other commonly used early and late promoters include those from SimianVirus 40 (SV 40) (Fiers, et al., Nature 273:113 (1973)), or other viralpromoters such as those derived from polyoma, Adenovirus 2, and bovinepapilloma virus. The controllable promoter, hMTII (Karin, et al., Nature299:797- 802 (1982)) may also be used. General aspects of mammalian cellhost system transformations have been described by Axel (U.S. Pat. No.4,399,216 issued Aug. 16, 1983). It now appears, that “enhancer” regionsare important in optimizing expression; these are, generally, sequencesfound upstream or downstream of the promoter region in non-coding DNAregions. Origins of replication may be obtained, if needed, from viralsources. However, integration into the chromosome is a common mechanismfor DNA replication in eukaryotes.

Although preferred host cells for expression of the DNA constructsinclude eukaryotic cells such as COS or CHO cells, other eukaryoticmicrobes may be used as hosts. Laboratory strains of Saccharomycescerevisiae, Baker's yeast, are most used although other strains such asSchizosaccharomyces pombe may be used. Vectors employing, for example,the 2π origin of replication of Broach, Meth. Enz. 101:307 (1983), orother yeast compatible origins of replications (see, for example,Stinchcomb et al., Nature 282:39 (1979)); Tschempe et al., Gene 10:157(1980); and Clarke et al., Meth. Enz. 101:300 (1983)) may be used.Control sequences for yeast vectors include promoters for the synthesisof glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 (1968);Holland et al., Biochemistry 17:4900 (1978)). Additional promoters knownin the art include the CMV promoter provided in the CDM8 vector (Toyamaand Okayama, FEBS 268:217-221 (1990); the promoter for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073(1980)), and those for other glycolytic enzymes. Other promoters, whichhave the additional advantage of transcription controlled by growthconditions are the promoter regions for alcohol dehydrogenase 2,isocytoechrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and enzymes responsible for maltose and galactoseutilization. It is also believed terminator sequences are desirable atthe 3′ end of the coding sequences. Such terminators are found in the 3′untranslated region following the coding sequences in yeast-derivedgenes.

Alternatively, prokaryotic cells may be used as hosts for expression.Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used. Commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, include such commonly usedpromoters as the beta-lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al., Nature 198: 1056 (1977)), the tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980))and the lambda derived PL promoter and N-gene ribosome binding site(Shimatake et al., Nature 292:128 (1981)).

The nucleotide sequences encoding the amino acid sequences correspondingto the CD28Ig and B7Ig fusion proteins, may be expressed in a variety ofsystems as set forth below. The cDNA may be excised by suitablerestriction enzymes and ligated into suitable prokaryotic or eukaryoticexpression vectors for such expression. Because CD28 receptors occur innature as dimers, it is believed that successful expression of theseproteins requires an expression system which permits these proteins toform as dimers. Truncated versions of these proteins (i.e. formed byintroduction of a stop codon into the sequence at a position upstream ofthe transmembrane region of the protein) appear not to be expressed. Theexpression of CD28 antigen in the form of a fusion protein permits dimerformation of the protein. Thus, expression of CD28 antigen as a fusionproduct is preferred in the present invention.

Sequences of the resulting fusion protein constructs are confirmed byDNA sequencing using known procedures, for example, as described bySanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977) as furtherdescribed by Messing et al., Nucleic Acids Res. 9:309 (1981) or by themethod of Maxam et al. Methods Enzyimol. 65 :499 (1980)).

Recovery of Protein Products

As noted above, the CD28 receptor is not readily expressed as a matureprotein using direct expression of DNA encoding the amino acid sequencecorresponding to the truncated protein. To enable homodimer formation,it is preferred that DNA encoding the amino acid sequence correspondingto the extracellular domain of CD28 and including the codons for asignal sequence such as oncostatin M in cells capable of appropriateprocessing, is fused with DNA encoding amino acids corresponding to theFc domain of a naturally dimeric protein. Purification of the fusionprotein products after secretion from the cells is thus facilitatedusing antibodies reactive with the anti-immunoglobulin portion of thefusion proteins. When secreted into the medium, the fusion proteinproduct is recovered using standard protein purification techniques, forexample by application to protein A columns.

In addition to the fusion proteins of the invention, monoclonalantibodies reactive with the B7 antigen and CD28 receptor, and reactivewith B7Ig and CD28Ig fusion proteins, may be produced by hybridomasprepared using known procedures, such as those introduced by Kohler andMilstein (see Kohier and Milstein, Nature, 256:495-97 (1975), andmodifications thereof, to regulate cellular interactions.

These techniques involve the use of an animal which is primed to producea particular antibody. The animal can be primed by injection of animmunogen (e.g. the B7Ig fusion protein) to elicit the desired immuneresponse, i.e. production of antibodies reactive with the ligand forCD28, the B7 antigen, from the primed animal. A primed animal is alsoone which is expressing a disease. Lymphocytes derived from the lymphnodes, spleens or peripheral blood of primed, diseased animals can beused to search for a particular antibody. The lymphocyte chromosomesencoding desired immunoglobulins are immortalized by fusing thelymphocytes with myeloma cells, generally in the presence of a fusingagent such as polyethylene glycol (PEG). Any of a number of myeloma celllines may be used as a fusion partner according to standard techniques;for example, the P3- NS1/1-Ag4-1, P3-x63-Ag8.653, Sp2/0-Agl4, or HL1-653myeloma lines. These mycloma lines are available from the ATCC,Rockville, Md.

The resulting cells, which include the desired hybridomas, are thengrown in a selective medium such as HAT medium, in which unfusedparental myeloma or lymphocyte cells eventually die. Only the hybridomacells survive and can be grown under limiting dilution conditions toobtain isolated clones. The supernatants of the hybridomas are screenedfor the presence of the desired specificity, e.g. by immunoassaytechniques using the B7Ig fusion protein that has been used forimmunization. Positive clones can then be subcloned under limitingdilution conditions, and the monoclonal antibody produced can beisolated.

Various conventional methods can be used for isolation and purificationof the monoclonal antibodies so as to obtain them free from otherproteins and contaminants. Commonly used methods for purifyingmonoclonal antibodies include ammonium sulfate precipitation, ionexchange chromatography, and affinity chromatography (see Zola et al.,in Monoclonal Hybridoma Antibodies: Techniques and Apolications, Rurell(ed.) pp. 51-52 (CRC Press, 1982)). Hybridomas produced according tothese methods can be propagated in vitro or in vivo (in ascites fluid)using techniques known in the art (see generally Fink et al.,Prog.Clin.Pathol., 9:121-33 (1984), FIG. 6-1 at p. 123).

Generally, the individual cell line may be propagated in vitro forexample, in laboratory culture vessels, and the culture mediumcontaining high concentrations of a single specific monoclonal antibodycan be harvested by decantation, filtration, or centrifugation.

In addition, fragments of these antibodies containing the active bindingregion of the extracellular domain of B7 or CD28 antigen, such as Fab,F(ab′)₂ and Fv fragments, may be produced. Such fragments can beproduced using techniques well established in the art (see e.g.Rousseaux et al., in Methods Enzymol, 121:663-69, Academic Press(1986)).

USES

General

The experiments described below in the Examples, suggest that the CD28receptor and its ligand, the B7 antigen, may function in vivo bymediating T cell interactions with other cells such as B cells. Thefunctional consequences of these interactions may be induced orinhibited using ligands that bind to the native CD28 receptor or the B7antigen.

It is expected that administration of the B7 antigen will result ineffects similar to the use of anti-CD28 monoclonal antibodies (mAbs)reactive with the CD28 receptor in vivo. Thus, because anti-CD28 mAbsmay exert either stimulatory or inhibitory effects on T cells,depending, in part, on the degree of crosslinking or “aggregation” ofthe CD28 receptor (Damle, J. Immunol. 140:1753-1761 (1988); Ledbetter etal., Blood 75(7):1531-1539 (1990)) it is expected that the B7 antigen,its fragments and derivatives, will act to stimulate or inhibit T cellsin a manner similar to the effects observed for an anti-CD28 monoclonalantibody, under similar conditions in vivo. For example, administrationof B7 antigen, e.g. as a soluble B7Ig fusion protein to react with CD28positive T cells, will bind the CD28 receptor on the T cells and resultin inhibition of the functional responses of T cells. Under conditionswhere T cell interactions are occurring as a result of contact between Tcells and B cells, binding of introduced B7 antigen in the form of afusion protein that binds to CD28 receptor on CD28 positive T cellsshould interfere, i.e. inhibit, the T cell interactions with B cells.Likewise, administration of the CD28 antigen, or its fragments andderivatives in vivo, for example in the form of a soluble CD28Ig fusionprotein, will result in binding of the soluble CD28Ig to B7 antigen,preventing the endogenous stimulation of CD28 receptor by B7 positivecells such as activated B cells, and inteffering with the interaction ofB7 positive cells with T cells.

Alternatively, based on the known effects of aggregating the CD28receptor, either by reacting T cells with immobilized ligand, or bycrosslinking as described by Ledbetter et al., Blood 75(7):1531-1539(1990)), the B7 antigen, and/or its fragments or derivatives, may beused to stimulate T cells, for example by inunobilizing B7 antigen orB7Ig fusion protein, for reacting with the T cells. The activated Tcells stimulated in this manner in vitro may be used in vivo in adoptivetherapy.

Therefore, the B7 antigen and/or fragments or derivatives of the antigenmay be used to react with T cells to regulate immune responses mediatedby functional T cell responses to stimulation of the CD28 receptor. TheB7 antigen may be presented for reaction with CD28 positive T cells invarious forms. Thus, in addition to employing activated B cellsexpressing the B7 antigen, the B7 antigen may be encapsulated, forexample in liposomes, or using cells that have been geneticallyengineered, for example using gene transfer, to express the antigen forstimulation of the CD28 receptor on T cells.

The CD28 receptor, and/or its fragments or derivatives, may also be usedto react with cells expressing the B7 antigen, such as B cells. Thisreaction will result in inhibition of T cell activation, and inhibitionof T cell dependent B cell responses, for example as a result ofinhibition of T cell cytokine production.

In an additional embodiment of the invention, other reagents, such asmolecules reactive with B7 antigen or the CD28 receptor are used toregulate T and/or B cell responses. For example, antibodies reactivewith the CD28Ig fusion proteins, and Fab fragments of CD28Ig, may beprepared using the CD28Ig fusion protein as immunogen, as describedabove.

These anti-CD28 antibodies may be screened to identify those capable ofinhibiting the binding of the B7 antigen to CD28 antigen. The antibodiesor antibody fragments such as Fab fragments may then be used to reactwith the T cells, for example, to inhibit CD28 positive T cellproliferation. The use of Fab fragments of the 9.3 monoclonal antibody,or Fab fragments of the anti-CD28Ig monoclonal antibodies as describedherein, is expected to prevent binding of CD28 receptor on T cells to B7antigen, for example on B cells. This will result in inhibition of thefunctional response of the T cells.

Similarly, anti-B7 monoclonal antibodies such as BB-1 mAb, or anti-B7Igmonoclonal antibodies prepared as described above using B7Ig fusionprotein as immunogen, may be used to react with B7 antigen positivecells such as B cells to inhibit B cell interaction via the B7 antigenwith CD28 positive T cells.

In another embodiment the B7 antigen may be used to identify additionalcompounds capable of regulating the interaction between the B7 antigenand the CD28 antigen. Such compounds may include soluble fragments ofthe B7 antigen or CD28 antigen or small naturally occurring moleculesthat can be used to react with B cells and/or T cells. For example,soluble fragments of the ligand for CD28 containing the extracellulardomain (e.g. amino acids 1-215) of the B7 antigen may be tested fortheir effects on T cell proliferation.

Uses In Vitro and In Vivo

In a method of the invention, the ligand for CD28, B7 antigen, is usedfor regulation of CD28 positive (CD28 ⁺) T cells. For example, the B7antigen is reacted with T cells in vitro to crosslink or aggregate theCD28 receptor, for example using CHO cells expressing B7 antigen, orimmobilizing B7 on a solid substrate, to produce activated T cells foradministration in vivo for use in adoptive therapy. In adoptive therapyT lymphocytes are taken from a patient and activated in vitro with anagent. The activated cells are then reinfused into the autologous donorto kill tumor cells (see Rosenberg et al., Science 223:1318-1321(1986)). The method can also be used to produce cytotoxic T cells usefulin adoptive therapy as described in copending U.S. patent applicationSer. No. 471,934, filed Jan. 25, 1990, incorporated by reference herein.

Alternatively, the ligand for CD28, its fragments or derivatives, may beintroduced in a suitable pharmaceutical carrier in vivo, i.e.administered into a human subject for treatment of pathologicalconditions such as immune system diseases or cancer. Introduction of theligand in vivo is expected to result in interference with T cell/B cellinteractions as a result of binding of the ligand to T cells. Theprevention of normal T cell/B cell contact may result in decreased Tcell activity, for example, decreased T cell proliferation.

In addition, administration of the B7 antigen in vivo is expected toresult in regulation of in vivo levels of cytokines, including, but notlimited to, interleukins, e.g. interleukin (“IL”)-2, IL-3, IL-4, IL-6,IL-8, growth factors including tumor growth factor (“TGF”), colonystimulating factor (“CSF”), interferons (“IFNs”), and tumor necrosisfactor (“TNF”) to promote desired effects in a subject. It isanticipated that ligands for CD28 such as B7Ig fusion proteins and Fabfragments may thus be used in place of cytokines such as IL-2 for thetreatment of cancers in vivo. For example, when the ligand for CD28 isintroduced in vivo it is available to react with CD28 antigen positive Tcells to mimic B cell contact resulting in increased production ofcytokines which in turn will interact with B cells.

Under some circumstances, as noted above, the effect of administrationof the B7 antigen, its fragments or derivatives in vivo is stimulatoryas a result of aggregation of the CD28 receptor. The T cells arestimulated resulting in an increase in the level of T cell cytokines,mimicking the effects of T cell/B cell contact on triggering of the CD28antigen on T cells. In other circumstances, inhibitory effects mayresult from blocking by the B7 antigen of the CD28 triggering resultingfrom T cell/B cell contact. For example, the B7 antigen may block T cellproliferation. Introduction of the B7 antigen in vivo will thus produceeffects on both T and B cell mediated immune responses. The ligand mayalso be administered to a subject in combination with the introductionof cytokines or other therapeutic reagents. Alternatively, for cancersassociated with the expression of B7 antigen, such as B7 lymphomas,carcinomas, and T cell leukemias, ligands reactive with the B7 antigen,such as anti-B7Ig monoclonal antibodies, may be used to inhibit thefunction of malignant B cells.

Because CD28 is involved in regulation of the production of severalcytokines, including TNF and gamma interferon (Lindsten et al., supra.(1989)), the ligand for CD28 of the invention may be useful for in vivoregulation of cytokine levels in response to the presence of infectiousagents. For example, the ligand for CD28 may be used to increaseantibacterial and antiviral resistance by stimulating tumor necrosisfactor (TNF) and lFN production. TNF production seems to play a role inantibacterial resistance at early stages of infection (Havell, J.Immunol. 143:2894-2899 (1990)). In addition, because herpes virusinfected cells are more susceptible to TNF-mediated lysis thanuninfected cells (Koff and Fann, Lymphokine Res. 5:215 (1986)), TNF mayplay a role in antiviral immunity.

Gamma interferon is also regulated by CD28 (Lindsten et al., supra).Because mRNAs for alpha and beta IFNs share potential regulatorysequences in their 3′ untranslated regions with cytokines regulated byCD28, levels of these cytokines may also be regulated by the ligand forCD28. Thus, the ligand for CD28 may be useful to treat viral diseasesresponsive to interferons (De Maeyer and De Maeyer-Guignard, inInterferons and Other Regulatory Cytokines, Wiley Publishers, New York(1988)). Following the same reasoning, the ligand for CD28 may also beused to substitute for alpha-IFN for the treatment of cancers, such ashairy cell leukemia, melanoma and renal cell carcinoma (Goldstein andLaszio, CA: a Cancer Journal for Clinicians 38:258-277 (1988)), genitalwarts and Kaposi's sarcoma.

In addition, B7Ig fusion proteins as described above may be used toregulate T cell proliferation. For example, the soluble CD28Ig and B7Igfusion proteins may be used to block T cell proliferation in graftversus host (GVH) disease which accompanies allogeneic bone marrowtransplantation. The CD28-mediated T cell proliferation pathway iscyclosporine-resistant, in contrast to proliferation driven by theCD3/Ti cell receptor complex (June et al., 1987, supra). Cyclosporine isrelatively ineffective as a treatment for GVH disease (Storb, Blood68:119-125 (1986)). GVH disease is thought to be mediated by Tlymphocytes which express CD28 antigen (Storb and Thomas, Immunol. Rev.88:215-238 (1985)). Thus, the B7 antigen in the form of B7Ig fusionprotein, or in combination with immunosuppressants such as cyclosporine,for blocking T cell proliferation in GVH disease. In addition, B7Igfusion protein may be used to crosslink the CD28 receptor, for exampleby contacting T cells with immobilized B7Ig fusion protein, to assist inrecovery of immune function after bone marrow transplantation bystimulating T cell proliferation.

The fusion proteins of the invention may be useful to regulategranulocyte macrophage colony stimulating factor (GM-CSF) levels fortreatment of cancers (Brandt et al., N. Eng. J. Med. 318:869-876(1988)), AIDS (Groopman et al., N. Eng. J. Med. 317:593-626 (1987)) andmyelodysplasia (Vadan-Raj et al., N. Eng. J. Med. 317:1545-1551 (1987)).

Regulation of T cell interactions by the methods of the invention maythus be used to treat pathological conditions such as autoimmunity,transplantation, infectious diseases and neoplasia.

In a preferred embodiment, the role of CD28-mediated adhesion in T celland B cell function was investigated using procedures used todemonstrate intercellular adhesion mediated by MHC class I (Norrnent etal., (1988) supra) and class II (Doyle and Strominger, (1987) supra)molecules with the CD8 and CD4 accessory molecules, respectively. TheCD28 antigen was expressed to high levels in Chinese hamster ovary (CHO)cells and the transfected cells were used to develop a CD28-mediatedcell adhesion assay, described infra. With this assay, an interactionbetween the CD28 antigen and its ligand expressed on activated Blymphocytes, the B7 antigen, was demonstrated. The CD28 antigen,expressed in CHO cells, was shown to mediate specific intracellularadhesion with human lymphoblastoid and leukemic B cell lines, and withactivated murine B cells. CD28-mediated adhesion was not dependent upondivalent cations. A mAb, BB-1, reactive with B7 antigen was shown toinhibit CD28-mediated adhesion. Transfected COS cells expressing the B7antigen were also shown to adhere to CD28 ⁺ CHO cells; this adhesion wasblocked by mAbs to CD28 receptor and B7 antigen. The specificrecognition by CD28 receptor of B7 antigen, indicated that B7 antigen isthe ligand for the CD28 antigen.

The results presented herein also demonstrate that antibodies reactivewith CD28 and B7 antigen specifically block helper T_(h)-mediatedimmunoglobulin production by allogeneic B cells, providing evidence ofthe role of CD28/B7 interactions in the collaboration between T and Bcells.

In additional preferred embodiments, B7Ig and CD28Ig fusion proteinswere constructed by fusing DNA encoding the extracellular domains of B7antigen or the CD28 receptor to DNA encoding portions of humanimmunoglobulin C gamma 1. These fusion proteins were used to furtherdemonstrate the interaction of the CD28 receptor and its ligand, the B7antigen.

The cell adhesion assay method of the invention permits identificationand isolation of ligands for target cell surface receptors mediatingintercellular adhesion, particularly divalent cation independentadhesion. The target receptor may be an antigen or other receptor onlymphocytes such as T or B cells, on monocytes, on microorganisms suchas viruses, or on parasites. The method is applicable for detection ofligand involved in ligand/receptor interactions where the affinity ofthe receptor for the ligand is low, such that interaction betweensoluble forms of the ligand and target receptor is difficult to detect.In such systems, adhesion interactions between other ligands andreceptors that are divalent cation dependent may “mask” otherinteractions between ligands for target receptors, such that theseinteractions are only observed when divalent cations are removed fromthe system.

The cell adhesion assay utilizes cells expressing target cell surfacereceptor and cells to be tested for the presence of ligand mediatingadhesion with the receptor. The cells expressing target receptor may becells that are transfected with the receptor of interest, such asChinese hamster ovary (CHO) or COS cells. The cells to be tested for thepresence of ligand are labeled, for example with ⁵¹Cr, using standardmethods and are incubated in suitable medium containing a divalentcation chelating reagent such as ethylenediamine tetraacetic acid (EDTA)or ethyleneglycol tetraacetic acid (EGTA). Alternatively, the assay maybe performed in medium that is free of divalent cations, or is renderedfree of divalent cations, using methods known in the art, for exampleusing ion chromatography. Use of a divalent cation chelating reagent orcation-free medium removes cation-dependent adhesion interactionspermitting detection of divalent cation-independent adhesioninteractions. The labeled test cells are then contacted with the cellsexpressing target receptor and the number of labeled cells bound to thecells expressing receptor is determined by measuring the label, forexample using a gamma counter. A suitable control for specificity ofadhesion can be used, such as a blocking antibody, which competes withthe ligand for binding to the target receptor.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of thedisclosure or the protection granted by Letters Patent hereon.

EXAMPLE 1

Identification of the Ligand For CD28 Receptor

If CD28 receptor antigen binds to a cell surface ligand, then cellsexpressing the ligand should adhere more readily to cells expressingCD28 receptor than to cells which do not. To test this, a cDNA cloneencoding CD28 under control of a highly active promoter (Aruffo andSeed, (1987) supra) together with a selectable marker (pSV2dhfr)(Mulligan and Berg, Science 209:1414-1422 (1980)) was transfected intodihydrofolate reductase (dhfr)-deficient CHO cells.

Cell Culture. T51, 1A2, 5E1, Daudi, Raji, Jijoye, CEM, Jurkat, HSB2,THP-1 and HL60 cells (Bristol-Myers Squibb Pharmaceutical ResearchInstitute, Seattle, Wash.) were cultured in complete RPMI™ medium (RPMI™containing 10 % fetal bovine serum (FBS), 100 U/ml penicillin and 100μg/ml streptomycin. Dhfr-deficient Chinese hamster ovary (CHO) cells(Urlaub and Chasin, Proc. Natl. Acad Sci., 77:4216-4220 (1980)) werecultured in Maintenance Medium (Ham's F12 Medium™ (GIBCO, Grand Island,N.Y.) supplemented with 10 % FBS, 0.15 mM L-proline, 100 U/ml penicillinand 100 μg/ml streptomycin). Dhfr-positive transfectants were selectedand cultured in Selective Medium (DMEM™, supplemented with 10 % FBS,0.15 mM L-proline, 100 U/ml penicillin and 100 μg/ml streptomycin).

Spleen B cells were purified from Balb/c mice by treatment of totalspleen cells with an anti-Thy 1.2 mAb (30H12) (Ledbetter and Herzenberg,Immunol. Rev. 47:361-389 (1979)) and baby rabbit complement. Theresulting preparations contained approximately 85 % B cells, as judgedby FACS^(R) analysis following staining with fluoresceinisothiothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin(TAGO). These cells were activated by treatment for 72 hrs with E. colilipopolysaccharide (LPS, List Biological Laboratories, Campbell, Calif.)at 10 μg/ml in complete RPMI™.

Monoclonal Antibodies. Monoclonal antibody (mAb) 9.3 (anti-CD28) (ATCCNo. HB 10271, Hansen et al., Immunogenetics 10:247-260 (1980)) waspurified from ascites before use. mAb 9.3 F(ab′)₂ fragments wereprepared as described by Parham, in J. Immunol. 131:2895-2902 (1983).Briefly, purified mAb 9.3 was digested with pepsin at pH 4.1 for 75 min.followed by passage over protein A SEPHAROSE™ (beaded agarose) to removeundigested mAb. A number of mAbs to B cell-associated antigens werescreened for their abilities to inhibit CD28-mediated adhesion. mAbs60.3 (CD18); 1FS (CD20); G29-5 (CD21); G28-7, HD39, and HD6 (CD22); HD50(CD23); KB61 (CD32); G28-1 (CD37); G28-10 (CD39); G28-5 (CD40); HERMES1(CD44); 9.4 (CD45); LB-2 (CD54) and 72F3 (CD71) have been previouslydescribed and characterized in International Conferences on HumanLeukocyte Differentiation Antigens I-Ill (Bernard et al., Eds.,Leukocyte Typing, Springer-Verlag, N.Y. (1984); Reinherz et al., Eds.,Leukocyte Typing II Vol. 2 N.Y. (1986); and McMichael et al., Eds.,Leukocyte Typing III Oxford Univ. Press, N.Y., (1987)). These mAbs werepurified before use by protein A SEPHAROSE™ (beaded agarose)chromatography or by salt precipitation and in exchange chromatography.δTA401 (Kuritani and Cooper, J. Exp. Med. 155:839-848 (1982))(Anti-IgD); 2C3 (Clark et al., (1986), supra) (anti-IgM); Nambi, H1DE,P10.1, W6/32 (Clark et al., (1986) supra; and Gilliland et al., HumanImmunology 25:269-289 (1989), anti-human class I); and HB1OA (Clark etal., (1986), supra, anti-MHC class II) were also purified before use.mAbs B43 (CD19); BL-40 (CD72); AD2, 1E9.28.1, and 7G2.2.11 (CD73);EBU-141, LN1 (CDw75); CRIS-1 (CD-76); 424/4A11, 424/3D9 (CD77) Leu 21,Ba, 1588, LO-panB-1, FN1, and FN4 (CDw78); and M9, G28-10, HuLyml0, 2-7,F2B2.6, 121, L26, HD77, NU-B1, BLAST-1, BB-1, anti-BL7, anti-HC2, andL23 were used as coded samples provided to participants in the FourthInternational Conference on Human Leukocyte Differentiation Antigens(Knapp, Ed., Leukocyte Typing IV, Oxford Univ. Press, N.Y. (1990). Thesewere used in ascites form. mAbs BB-1 and LB-1 (Yokochi et al., (1981),supra) were also purified from ascites before use. Anti-integrinreceptor mAbs P3E3, P4C2, P4G9 (Wayner et al., J., Cell. Biol.109:1321-1330 (1989)) were used as hybridoma culture supernatants.

Immunostaining Techniques. For indirect immunofluorescence, cells wereincubated with mAbs at 10 μg/ml in complete RPMI™ for 1 hr at 4° C. mAbbinding was detected with a FITC-conjugated goat anti-mouseimmunoglobulin second step reagent. For direct binding experiments, mAbs9.3 and BB-1 were directly conjugated with FITC as described by Godingin Monoclonal Antibodies: Principles and Practices Academic Press,Orlando, FL (1983), and were added at saturating concentrations incomplete RPMI™ for 1 hr at 4° C. Non-specific binding of FITC-conjugatedmAbs was measured by adding the FITC conjugate following antigenpre-blocking (20-30 min at 4° C.) with unlabeled mAb9.3 or BB-1.Immunohistological detection of adherent lymphoblastoid cells wasachieved using the horseradish peroxidase (HRP) method described byHelistrom et al., J. Immunol. 127:157-160 (1981).

Plasmids and Transfections. cDNA clones encoding the amino acidsequences corresponding to T cell antigens CD4, CD5 and CD28 in theexpression vector pπH3M (Aruffo and Seed (1987), supra)), were providedby Drs. S. Aruffo and B. Seed, Massachusetts General Hospital, Boston,Mass. An expressible cDNA clone in the vector CDM8 encoding the aminoacid sequence corresponding to B7 antigen (Freeman et al., J. Immunol.143:2714-2722 (1989)) was provided by Dr. Gordon Freeman (Dana FarberCancer Institute, Boston, Mass.).

Dhfr-deficient CHO cells were co-transfected with a mixture of 9 μg ofplasmid πH3M-CD28 (Aruffo and Seed, (1987) supra) and 3 μg of plasmidpSV2dhfr (Mulligan and Berg, (1980), supra) using the calcium phosphatetechnique (Graham and Van Der Eb, Virology 52:456-467 (1973)).Dhfr-positive colonies were isolated and grown in Selective Mediumcontaining increasing amounts of methotrexate (Sigma Chemical Co., St.Louis, Mo.). Cells resistant to 10 nM methotrexate were collected byincubation in PBS containing 10 mM EDTA, stained for presence of theCD28 receptor by indirect immuofluorescence, and separated by FACS^(R)into CD28-positive (CD28 ⁺) and CD28-negative (CD28) populations. Bothpopulations were again cultured in Selective Medium containingincreasing concentrations of methotrexate to 1 μM, stained for the CD28antigen and again sorted into CD28 ⁺ and CD28 ⁻ populations.

COS cells were transfected with B7, CD4 or CD5 cDNAs as described byMalik et at., Molecular and Cellular Biology 9:2847-2853 (1989).Forty-eight to seventy-two hours after transfection, cells werecollected by incubation in PBS containing 10 mM EDTA, and used for flowcytometry analysis or in CD28-mediated adhesion assays as described,infra.

Cell lines expressing high (CD28 ⁺) and low (CD28 ⁻) levels of the CD28receptor were isolated from amplified populations by FACS^(R) sortingfollowing indirect immunostaining with mAb 9.3. After two rounds ofFACS^(R) selection, the CD28 ⁺ population stained uniformly positivewith FITC-conjugated mAb 9.3 (mean channel, 116 in linear fluorescenceunits), while the CD28 ⁻ population stained no brighter (mean channel,3.9) than unstained cells (mean channel, 3.7). Staining by CD28 ⁺ CHOcells was approximately ten-fold brighter thanphytohemagglutin-stimulated T cells (mean channel, 11.3). The CD28 ⁺ andCD28 ⁻ populations stably maintained their phenotypes after more than 6months of continuous culture in Selective Medium containing 1 μM ofmethotrexate.

Cell Adhesion Assay for a Ligand For CD28

An adhesion assay to detect differential binding to CD28 ⁺ and CD28 ⁻CHO cells by cells expressing a ligand for CD28 was developed. Since mAb9.3 has been shown to inhibit mixed lymphocyte reactions using Blymphoblastoid cells lines as a source of alloantigen (Damle et al.,(1981) supra; and Lesslauer et al., Eur. J. Immunol. 16:1289-1296(1986)) B lymphoblastoid cell lines were initially tested forCD28-mediated adhesion.

CD28-Mediated Adhesion Assay

Cells to be tested for adhesion were labeled with ⁵¹Cr (0.2-1 mCi) tospecific activities of 0.2-2 cpm/cell. A mouse mAb having irrelevantspecificity, mAb W1, directed against human breast carcinoma-associatedmucin, (Linsley et al., Cancer Res. 46:5444-5450 (1986)), was added tothe labeling reaction to a final concentration of 100 μg/ml to saturateFc receptors. Labeled and washed cells were preincubated in completeRPMI™ containing 10 μg/ml of mAb W1, and unless otherwise indicated, 10mM EDTA. mAb 9.3 or mAb 9.3 F(ab′)₂ was added to some samples at 10μg/ml, for approximately 1 hr at 23° C.

Labeled cells (1-10×10⁶/well in a volume of 0.2 ml complete RPMI™,containing EDTA and mAbs, where indicated) were then added to the CHOmonolayers. Adhesion was initiated by centrifugation in a plate carrier(1,000 rpm, in a Sorvall HB1OOO rotor, approximately 210 X g) for 3 minat 4° C. Plates were then incubated at 37° C. for 1 hr.

Reactions were terminated by aspirating unbound cells and washing fivetimes with cold, complete RPMI™. Monolayers were solubilized by additionof 0.5 N NaOH, and radioactivity was measured in a gamma counter. Formost experiments, numbers of bound cells were calculated by dividingtotal bound radioactivity (cpm) by the specific activity (cpm/cell) oflabeled cells. When COS cells were used, their viability at the end ofthe experiment was generally less than 50 %, so specific activitycalculations were less accurate. Therefore, for COS cells results areexpressed as cpm bound.

In pilot experiments, T51 lymphoblastoid cells were found to adhere moreto CD28 ⁺ CHO cells, than to CD28 ⁻]CHO cells. Fuithennore, adhesion ofT51 cells to CD28 ⁼ CHO cells was partially blocked by mAb 9.3, whileadhesion to CD28 ⁻ CHO cells was not consistently affected. Adhesion wasnot affected by control mAb L6 (ATCC No. KB 8677, Hellstrom et al.,Cancer Res. 46:3917-3923 (1986)), which is of the same isotype as mAb9.3 (IgG2a). These experiments suggested that T51 cells adheredspecifically to CD28 ⁺ CHO cells. Since blocking of adhesion by mAb 9.3was incomplete, ways to increase the specificity of the CD28 adhesionassay were explored.

The effects of divalent cation depletion on T51 cell adhesion to CD28 ⁺and CD28 ⁻ CHO cells were examined. Preliminary experiments showed thatEDTA treatment caused loss of CHO cells during washing, so the CHO cellmonolayers were fixed with paraformaldehyde prior to EDTA treatment.Fixation did not significantly affect CD28-mediated adhesion by T51cells either in the presence or absence of mAb 9.3. Monolayers of CD28 ⁺and CD28 ⁻ CHO cells (1 to 1.2×10⁵/cm² in 48 well plastic dishes) werefixed in 0.5 % paraformaldehyde for 20 min at 23° C., washed and blockedin Complete RPMI™ for 1 hr, then pre-incubated with or without mAb 9.3or mAb 9.3 F(ab')₂ at 10 μg/ml in Complete RPMI™ for 1 hr at 37° C. T51cells were labeled with ⁵¹Cr, preincubated with or without 10mM EDTA,added to CHO cells and cellular adhesion was measured. The results arepresented in FIG. 1. Mean and standard deviation (error bars) are shownfor three replicate determinations.

The specificity of CD28-mediated adhesion was greatly increased in thepresence of EDTA (FIG. 1). Adhesion to CD28 ⁺ cells in the presence ofEDTA was 17-fold greater than to CD28 ⁻ cells in the presence of EDTA,compared with 5.5-fold greater in its absence. Adhesion to CD28 ⁺ cellsin the presence of mAb 9.3 plus EDTA was reduced by 93 %, compared with62 % in the presence of mAb alone. CD28-mediated adhesion of T51 cellsin the presence of EDTA could also be seen quite clearly by microscopicexamination following immunohistological staining of T51 cells. Cellularadhesion between unlabeled T51 cells and CD28 ⁺ or CD28 ⁻ CHO cells wasdetermined in the presence of 10 mM EDTA as described above. AdherentT51 cells were stained with biotinylated anti-human Class II Ab, HB1Oa,fixed with 0.2 % glutaraldehyde and visualized by sequential incubationwith avidin-conjugated HRP (Vector Laboratories, Inc., Burlingame,Calif.) and diaminobenzidine solution (Hellstrom and Hellstrom, J.Immunol. 127:157-160 (1981)). The results of staining are shown in FIG.2. A similar, but slightly less significant increase in adhesionspecificity, was also observed in the presence of the calcium-specificchelator, EGTA.

The Ligand For CD28 is a B Cell Activation Marker

The increased specificity of CD28-mediated adhesion in EDTA made itpossible to more readily detect adhesion by cells other than T51. Anumber of additional cell lines were tested, including threelymphoblastoid lines (T51, 1A2, and 5E1); four Burkett's Lymphoma lines(Daudi, Raji, Jijoye, and Namalwa); one acute lymphoblastic (B cell)leukemia (REH); three T cell leukemias (CEM, Jurkat and HSB2); and twomonocytic leukemias (THP-1 and HL60). As a source of primary B cells,murine splenic B cells, before and after activation with LPS, weretested. All cells were tested for adhesion to both CD28 ⁺ and CD28 ⁻ CHOcells, in the absence and presence of MAb 9.3. The cells were labeledwith ⁵¹Cr and CD28-mediated adhesion was measured as described above.Three representative experiments showing adhesion to CD28 ⁺ CHO cellsare shown in FIG. 3. Inhibition by mAb 9.3 is shown as an indicator ofspecificity; in most cases, adhesion measured in the presence of mAb 9.3was approximately equal to adhesion to CD28 ⁺ cells.

CD28-specific adhesion (i.e., adhesion being greater than 70 %inhibitable by mAb 9.3), was observed with T51, 5E1, Raji, and Jijoyecells. Daudi cells also showed specific adhesion, although to a lesserextent. Other cell lines did not show specific CD28-mediated adhesion,although some (e.g., Namalwa) showed relatively high non-specificadhesion. Primary mouse splenic B cells did not show CD28-mediatedadhesion, but acquired the ability to adhere following activation withLPS. In other experiments, six additional lymphoblastoid lines showedCD28-mediated adhesion, while the U937 cell line, unstimulated humantonsil B cells, and phytohemagglutinin stimulated T cells did not showadhesion. These experiments indicate that a ligand for CD28 is found onthe cell surface of activated B cells of human or mouse origin.

CD28-Mediated Adhesion is Specifically Blocked by a mAb (BB-1) to B7Antigen

In initial attempts to define B cell molecules involved in CD28-mediatedadhesion, adhesion by lymphoblastoid cell lines having mutations inother known cellular adhesion molecules was measured using the adhesionassay described above. The 616 lymphoblastoid line (MHC classII-deficient) (Gladstone and Pious, Nature 271:459-461 (1978)) bound toCD28 ⁺ CHO cells equally well or better than parental T51 cells.Likewise, a CD 18-deficient cell line derived from a patient withleukocyte adhesion deficiency (Gambaro cells) (Beatty et al., Lancet1:535-537 (1984)) also adhered specifically to CD28. Thus, MHC class IIand CD18 molecules do not mediate adhesion to CD28.

A panel of mAbs to B cell surface antigens were then tested for theirability to inhibit CD28-mediated adhesion of T51 cells. For theseexperiments, a total of 57 mAbs reactive with T51 cells were tested,including mAbs to the B cell-associated antigens CD19, CD20, CD21, CD22,CD23, CD37, CD39, CD40, CD71, CD72, CD73, CDw75, CD76, CD77, CDw78, 1gM,and IgD; other non-lineage-restricted antigens CD18, CD32, CD45, CD54,and CD7l; CD44 and another integrin; MHC class I and class II antigens;and 30 unclustered B cell associated antigens. In addition to these,many other mAbs which did not react with T51 by FACS^(R) analysis weretested. Initial screening experiments were carried out in the absence ofEDTA, and any mAbs which blocked adhesion were subsequently retested inthe presence and absence of EDTA. Of these mAbs, only those directedagainst MHC class I molecules (Namb1, H1DE, P10.1, W6/32), and one to anunclustered B cell antigen (BB-l), originally described as a B cellactivation marker (Yokochi et al., (1981) supra) were consistently ableto block CD28-mediated adhesion by greater than 30 %.

The dose-dependence of adhesion inhibition by the anti-Class I mAb,H1DE, by BB-1 and by 9.3 were compared in the presence of EDTA in theexperiment shown in FIG. 4. Jijoye cells were labeled with ⁵¹Cr andallowed to adhere to CD28+ CHO cells in the presence of 10 mM EDTA asdescribed above. Adhesion measured in the presence of the indicatedamounts of mAbs 9.3, H1DE (anti-human class I MHC, Gaur et al.,Immunogenetics 27:356-361 (1988)), or mAb BB-1 is expressed as apercentage of maximal adhesion measured in the absence of mAb (45,000cells bound). mAb 9.3 was most effective at blocking, but mAb BB- 1 wasable to block approximately 60 % of adhesion at concentrations less than1 μg/ml. mAb H1DE also partially blocked adhesion at all concentrationstested. When EDTA was omitted from the adhesion assay, blocking by classI mAbs was consistently less, and required higher mAb concentrations,than mAbs 9.3 or BB-1.

Binding of mAb BB-l by Different Cells Correlates With CD28-SpecificAdhesion

To investigate the roles of molecules recognized by anti-class I andBB-1 mAbs in CD28-mediated adhesion, levels of these antigens on certainof the cell lines tested for CD28-specific adhesion in FIG. 3 werecompared. Cells were analyzed by FACS^(R) following indirectimmunofluorescence staining with mAbs H1DE and BB-1. Cell lines 1A2,Namalwa, REH and HL60 (which did not adhere specifically to CD28) allbound high levels of mAb H1DE, whereas Daudi cells (which did adhere)did not show detectable binding. Therefore, a direct correlation betweenCD28-mediated adhesion and expression of class I antigens was notobserved. On the other hand, these experiments suggested a correlationbetween adhesion to CD28 and staining by mAb BB-1.

To confirm this correlation, cell lines examined for CD28-mediatedadhesion in FIG. 3 were tested for staining by direct immunofluorescenceusing FITC-conjugated niAb BB 1 (Table 1). Cell lines were incubatedwith no mAb or with FITC-conjugated mAb BB-1 with or withoutpreincubation with cold (unlabeled) BB-1 mAb. Values shown in Table 1represent mean fluorescence in linear units. All of the cell lines whichadhered specifically to CD28 receptor (FIG. 3) bound higher levels ofthe FITC-conjugate than those which did not adhere specifically. Antigenspecificity was demonstrated in all cases by the ability of unlabeledmAb BB-1 to compete for binding of the FITC-conjugate.

TABLE 1 CELLS WHICH ADHERE TO CD28 ANTIGEN ALSO BIND MAb BB-1 FITC-BB-1SPECIFIC⁴ LINE CELL TYPE¹ NO MAb −COLD +COLD BINDING Positive for CD28Adhesion² T51 B-LCL 2.3 16.4 3.0 13.4 5E1 B-LCL 2.1 13.0 2.4 10.6 JijoyeBL 2.3 17.8 2.8 15.0 Raji BL 2.1 7.1 2.8 4.3 Daudi BL 2.1 6.4 2.8 3.6Negative for CD28 Adhesion³ 1A2 B-LCL 2.1 4.5 4.3 <1 Namalwa BL 2.2 3.82.2 1.6 REH B-ALL 2.0 2.1 2.0 <1 CEM T-ALL 2.3 2.0 1.9 <1 Jurkat T-ALL2.2 2.2 2.0 <1 HSB2 T-ALL 2.1 2.3 2.3 <1 HL60 AML 2.3 3.1 3.1 <1 THP-1AML 2.3 3.1 3.0 <1 ¹B-LCL = B-lymphoblastoid cell line BL = Burkett'slymphoma B-ALL = B cell-derived acute lymphoblastoid leukemia T-ALL = Tcell-derived acute lymphoblastoid leukemia AML = Acute Monocyticleukemia ²Positive for CD28 adhesion => 70% inhibition of adhesion byMAb 9.3 ³Negative for CD28 Adhesion =< 70% inhibition of adhesion by MAb9.3. ⁴Specific binding = (FITC-BB-1 + cold) subtracted from (FITC-BB-1 −cold).COS Cells Expressing the B7 Antigen Adhere Specifically to CD28

The role of the B7 antigen recognized by mAb BB-1 in CD28-mediatedadhesion was investigated using a cDNA clone isolated and sequenced byFreeman et al. as described in J. Immunol. 143:2714-2722 (1989). COScells were transfected with an expression vector containing the cDNAclone encoding the B7 antigen, as described by Freeman et al., (1989),supra as described above. Forty-eight hours later, transfected COS cellswere removed from their dishes by incubation in PBS containing 10 mMEDTA, and were labeled with ⁵¹Cr. Cells were shown to express B7 antigenby FACS^(R) analysis following indirect staining with mAb BB-1 asreported by Freeman et al, supra. Adhesion between B7 transfected COScells and CD28 ⁺ or CD28 ⁻ CHO cells was then measured in the presenceof 10 mM EDTA as described above. Where indicated, adhesion was measuredin the presence of mAbs 9.3 or BB-1 (10 μg/ml). As shown in FIG. 5,B7/BB-1-transfected COS cells adhered readily to CD28 ⁺ CHO cells;adhesion was completely blocked by both mAbs 9.3 and BB-1. No adhesionto CD28 ⁻ CHO cells was detected. This experiment was repeated fivetimes with identical results.

In other experiments, adhesion was not blocked by non-reactive, isotypematched controls, mAb W5 (1gM) (Linsley, (1986) supra) and mAb L6(IgG2A) (Helistrom et al., (1986) supra), or by mAb H1DE, which reactswith class I antigens on COS cells. CD28-mediated adhesion by B7transfected cells could also be clearly seen by microscopic examinationof the CHO cell monolayers after the assay. When COS cells weretransfected with expressible CD4 or CD5 cDNA clones, no CD28-mediatedadhesion was detected. Expression of CD4 and CD5 was confirmed byFACS^(R) analysis following immunofluorescent staining. When EDTA wasomitted from the assay, adhesion measured with CD5-transfected COS cellswas greatly increased but not inhibited by mAb 9.3. In contrast,adhesion by B7 transfected COS cells under these conditions was stillpartially blocked (approximately 40 %) by mAb 9.3. Thus, transfection ofB7 into COS cells confers the ability on the cells to adherespecifically to CD28 receptor.

The above assay for intracellular adhesion mediated by the CD28receptor, described above, demonstrated CD28-mediated adhesion byseveral lymphoblastoid and leukemic B cell lines, and by primary murinespleen cells following activation with LPS. These results indicate thepresence of a natural ligand for the CD28 receptor on the cell surfaceof some activated B lymphocytes.

Several lines of evidence show that the B cell molecule which interactedwith the CD28 receptor is the B7 antigen. mAb BB-1 was identified from apanel of mAbs as the mAb which most significantly inhibitedCD28-mediated adhesion. Furthermore, a correlation was observed betweenthe presence of B7 antigen and CD28-mediated adhesion (Table 1).Finally, COS cells transfected with B7 cDNA demonstrated CD28-mediatedadhesion. Taken together, these observations provide strong evidencethat B7 antigen is a ligand for CD28 receptor. Because both CD28 (Aruffoand Seed, (1987) supra) and B7 (Freeman et al., (1989) supra) aremembers of the immunoglobulin superfamily, their interaction representsanother example of heterophilic recognition between members of this genefamily (Williams and Barclay (1988), supra).

CD28-mediated adhesion differs in several respects from other celladhesion systems as shown in the above results. CD28-mediated adhesionwas not blocked by mAbs to other adhesion molecules, including mAbs toICAM-1 (LB-2), MHC class II (HB10a) CD18 (60.3), CD44 (HERMES-1 homingreceptor), and an integrin (P3E3, P4C2, P4G9). CD28-mediated adhesionwas also resistant to EDTA and EGTA, indicating that this system doesnot require divalent cations, in contrast to integrins (Kishimoto etal., Adv. Immunol. 46:149-182 (1989)) and some homing receptors(Stoolman, Cell 56:907-910 (1989)) which require divalent cations. Inthe system described herein, in which CD28 receptor was expressed tohigh levels relative to those on activated T cells, it was sometimesdifficult to measure CD28-mediated adhesion because of cation-dependent“background” adhesion (i.e., that not blocked by MAb 9.3, see FIG. 1).Preliminary experiments suggest that background adhesion in the absenceof EDTA was also blocked by MAb 60.3, which inhibits adhesion mediatedby LFA-1 (Pohinian et al., J. lmmunol. 136:4548-4553 (1986)). Even underoptimal conditions, some cells (such as Namalwa, see FIG. 3) showedsignificant non-CD28 dependent adhesion to CHO cells. Non-CD28 mediatedadhesion systems may also be responsible for the incomplete blockage bymAb BB-1 of B cell adhesion (FIG. 4). That this mAb is more effective atblocking adhesion by transfected COS cells (FIG. 5) may indicate thatnon-CD28 mediated systems are less effective in COS cells.

Finally, CD28-mediated adhesion appears more restricted in its cellulardistribution to T and B cells as compared to other adhesion molecules.CD28 receptor is primarily expressed by cells of the T lymphocytelineage. The B7 antigen is primarily expressed by cells of the Blymphocyte lineage. Consistent with this distribution, the ligand forCD28 was only detected on cells of B lymphocyte lineage. Thus, availabledata suggest that CD28 mediates adhesion mainly between T cells and Bcells. However, since CD28 expression has been detected on plasma cells(Kozbor et al., J. Immunol 138:4128-4132 (1987)) and B7 on cells ofother lineages, such as monocytes (Freeman et al., (1989) supra), it ispossible that other cell types may also employ this system.

Many adhesion molecules are known to mediate T cell-B cell interactionsduring an immune response and the levels of several of these, includingCD28 and B7 antigen, have been reported to increase followingactivation. Increased levels of these molecules may help explain whyactivated B cells are more effective at stimulating antigen-specific Tcell proliferation than are resting B cells. Because the B7 antigen isnot expressed on resting B cells, CD28-mediated adhesion may play a rolein maintaining or amplifying the immune response, rather than initiatingit. Such a role is also consistent with the function of CD28 inregulating lymphokine and cytokine levels (Thompson et al., (1989),supra; and Lindsten et al., (1989), supra).

EXAMPLE 2

Characterization of Interaction Between CD28 Receptor and B7 Antigen

This example used alloantigen-driven maturation of B cells as a modelsystem to demonstrate the involvement of the CD28 receptor on thesurface of major histocompatibility complex (MHC) class IIantigen-reactive CD4 positive T helper (T_(h)) cells and antigenpresenting B cells during the T_(h)-B cell cognate interaction leadingto B cell differentiation into immunoglobulin-secreting cells (IgSC).

Cognate interaction between CD4 ⁺ T_(h) and antigen-presenting B cellsresults in the activation and differentiation of both cell typesconsequently leading to the development of immunoglobulin-secretingcells (Moller (Ed) Immunol Rev. 99:1 (1987), supra). Allogenic MLRoffers an ideal system to analyze cognate T_(h)-B cell interactionbecause alloantigen-specific CD4 ⁺ Tb induce both the activation anddifferentiation of alloantigen-bearing B cells into immunoglobulinsecreting cells (Chiorazzi et al., Immunol Rev. 45:219 (1979); Kotzin etal., J. Immunol. 127:931 (1981); Friedman et al., J. Immunol. 129:2541(1982); Goldberg et al., J. Immunol. 135:1012 (1985); and Crow et al.,J. Exp. Med. 164:1760 (1986)). The involvement of the CD28 receptor onT_(h) cells and its ligand B7 during the activation of T_(h) and B cellsin the allogeneic MLR was first examined using murine mAb directed atthese molecules. Culture medium. Complete culture medium (CM) consistedof RPMI™ 1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with100 U/ml of penicillin G, 100 μg/ml of streptomycin, 2mM L-glutamine,5×10⁻⁵ M 2-ME, and 10 % FBS (Irvine Scientific). Cells and mAbs.EBV-transformed B cell lines CESS (HLA-AS1, A3; B5, B17; DR7), JIJOYE,and SKW6.4 (HLA-Ala; B27, B51; DR7), were obtained from the ATCC.EBV-transfonned B cell lines ARENT (HLA-A2; B38, B39, DRw6) and MSAB(HLA A1, A2; B57; DR7) were provided by Dr. E. G. Engleman, StanfordUniversity School of Medicine, Stanford, Calif. Hybridomas OKT4 (IgGanti-CD4), OKT8 (IgG anti-CD8) and HNK1 (1gM anti-CD57) were obtainedfrom the ATCC and ascitic fluids from these hybridomas were generated inpristane-primed BALB/c mice. Production and characterization ofanti-CD28 mAb 9.3 (IgG2a) has been described by Ledhetter et al., J.Immunol. 135:2331 (1985); Hara et al., J. Exp. Med. 161:1513 (1985) andMartin et al., J. Immunol. 136:3282 (1986), incorporated by referenceherein. mAb 4H9 (IgG2a anti-CD7) as described by Damle and Doyle, J.Immunol 143:1761 (1989), incorporated by reference herein, was providedby Dr. Engleman and mAb anti-B7 antibody (BB1; 1gM) as described byTokochi et al., J. Immunol. 128:823 (1981), incorporated by referenceherein, was provided by Dr. E. Clark, University of Washington, Seattle,Wash.

Peripheral blood mononuclear cells (PBMC) from healthy donors wereseparated into T and non-T cells using a sheep erythrocyte rosettingtechnique, and T cells were separated by panning into CD4 ⁺ subset andfurther into CD4 ⁺CD45RA - CD45RO⁺ memory subpopulation as described byDamle et al., J. Immunol. 139:1501 (1987), incorporated by referenceherein.

Proliferative responses of T cells. To examine the effect of anti-CD28and anti-B7 mAbs on the proliferative responses of T cells,fifty-thousand CD4 ⁺CD45RO⁺ T cells were stimulated by culturing with1×10⁴ irradiated (8000 rad from a ¹³⁷Cs source) EBV-transformedallogenic B cells (or 2.5×10⁴ non-T cells) in 0.2 ml of CM inround-bottom microtiter wells in a humidified 5 % CO₂ and 95 % airatmosphere in the presence of 10 μg/ml of mAb reactive with CD7, CD28,CD57 or B7 antigen. CD4 ⁺CD45RO⁺ T cells also were also independentlystimulated with 100 μg/ml of soluble purified protein derivative oftuberculin (PPD, Gonnough Laboratories, Willowdale, Ontario, Canada) inthe presence of 1×10⁴ irradiated (3000 rad) autologous non-T cells inthe presence of the above mAbs. Triplicate cultures were pulsed with 1μCi/well=37 kBq/well of [³H]dThd (6.7 Ci/mmol, NEN, Boston, Mass.) for16 h before harvesting of cells for measurement of radiolabelincorporation into newly synthesized DNA. The results are expressed ascpm±SEM. Proliferative responses were examined on day 7 of culture.EBV-transformed B cell lines were used as stimulator cells in theseexperiments because these B cells exhibit various features of activatedB cells such as the expression of high levels of MHC class II and B7molecules (Freeman et al., J. lmmunol. 139:3260 (1987); and Yokochi etal., J. Immunol. 128:823 (1981)).

FIG. 6 shows the results of these experiments. The presence of anti-CD28mAb (9.3 IgG2a) but not that of isotype-matched anti-CD7 mAb (4H9,IgG2a) consistently inhibited the MLR proliferative response of CD4 ⁺ Tcells to allogeneic B cells. Similarly, the addition of anti-B7 mAb(BB1; 1gM) but not that of isotype-matched anti-CD57 HNK1; 1gM) to theallogeneic MLR resulted in the inhibition of T cell proliferation. Theinhibitory effects of anti-CD28 mAb 9.3 on the MLR responses of T cellsare consistent with previous observations reported by Damle et al., J.Immunol. 120:1753 (1988) and Damle et al., Proc. Natl. Acad. Sci. USA78:5096 (1981). Similar to the allogeneic MLR, proliferative response ofCD4 ⁺ T cells to soluble Ag PPD presented by autologous non-T cells wasalso inhibited by anti-CD28 and anti-B7 mAb. Although both anti-CD28 mAb9.3 (IgG2a) and anti-B7 mAb, BB1 (1gM) inhibited the allogeneic MLR andthe soluble antigen-induced proliferative responses, anti-CD28-mediatedinhibition was always stronger than that by anti-B7 for all theresponder-stimulator combinations examined. These observations are alsoconsistent with the weaker ability of anti-B7 mAb to block theCD28-mediated adhesion to B7 ⁺ B cells as described above.

T cell-induced Immunoglobulin (Ig) production by B cells

To further examine the roles of CD28 and B7 during cognate T_(h)-Binteractions, two EBV-transformed B cells lines, IgG-secreting DR7 ⁺CESS and 1gM-secreting DR7 ⁺ SKW were used. When appropriatelystimulated, both these B cells lines significantly increase theirproduction of the respective Ig isotype. First, the effects ofDR7-specific CD4 ⁺ CD45RO⁺ T_(h) line on the Ig production of both CESSand SKW B cells was examined. DR7-primed CD4 ⁺ T_(h) cells were derivedfrom the allogeneic MLC consisting of responder CD4 ⁺ CD45RO⁺ T cells(HLA-A26, A29; B7, B55; DR9, DR10) and irradiated MSAB (DR7 ⁺ ) B cellsas stimulator cells as described by Damle et al., J. Immunol, 133:1235(1984), incorporated by reference herein. The isolation of resting CD4 ⁺CD45RO⁺ T cells and that of DR7-primed CD4 ⁺ CD45RO⁺ T lymphoblastsusing discontinuous Percoll density gradient centrifugation was also asdescribed by Damle, supra (1984). These DR7-primed CD4 ⁺ T_(h) cellswere continuously propagated in the presence of irradiated MSAB B cellsand 50 U/ml of IL-2. Prior to their functional analysis, viableDR7-prirned T_(h) cells were isolated by Ficoll-Hypaque gradientcentrifugation and maintained overnight in CM without DR7 ⁺ feeder cellsor IL-2, after which immunoglobulin secreted in the cell-free supematant(SN) was quantitated using a solid-phase ELISA.

To examine the effect of Tb cells on Ig production, by both CESS and SKWB cells 2×10⁴-2.5×10⁴ cells from HLA-DR7 ⁺ EBV-transformed B cell lines,1gM-producing SKW or IgG-producing CESS were cultured with varyingnumbers of DR7-primed CD4 ⁺CD45RO⁺ T_(h) cells for 96 h after whichcell-free SN from these cultures were collected and assayed for thequantitation of 1gM (SKW cultures) or IgG (CESS cultures) usingsolid-phase ELISA. Exogenous IL-6 (1-100 U/ml) induced Ig production bythese B cells was also used as a positive control to monitor thenon-cognate Ig production by these B cell lines. Ig production byfreshly isolated resting CD4 ⁺ CD45RO⁺ T_(h) cells (autologous to theDRt-primed CD4 ⁺ T_(h) cells) was also simultaneously examined as acontrol for DR7-primed CD4 ⁺ T_(h) cells.

Ig quantitation. IgG or 1gM in culture SN were measured usingsolid-phase ELISA as described by Volkman et al., Proc. Natl. Acad. Sci.USA 78:2528 (1981), incorporated by reference herein. Briefly, 96-wellflat-bottom microtiter ELISA plates (Corning, Corning N.Y.) were coatedwith 200 μl/well of sodium carbonate buffer (pH 9.6) containing 10 μg/mlof affinity-purified goat anti-human Ig or 1gM Ab (Tago, Burlingame,Calif.) incubated overnight at 4° C., and then washed with PBS and wellswere further blocked with 2 % BSA in PBS (BSA-PBS). Samples to beassayed were added at appropriate dilution to these wells and incubatedwith 200 μl/well of 1:1000 dilution of horseradish peroxidase(HRP)-conjugated F(ab′)₂ fraction of affinity-purified goat anti-humanIgG or 1gM Ab (Tago). The plates were then washed, and 100 μl/well ofo-phenylenediamine (Sigma, St. Louis, Mo.) solution (0.6 μg/ml incitrate-phosphate buffer with pH 5.5 and 0.045 % hydrogen peroxide).Color development was stopped with 2N sulfuric acid. Absorbance at 490nm was measured with an automated ELISA plate reader. Test and controlsamples were run in triplicate and the values of absorbance werecompared to those obtained with known IgG or 1gM standards runsimultaneously with the SN samples to generate the standard curve usingwhich the concentrations of Ig in culture SN were quantitated. Data areexpressed as ng/ml of Ig±SEM of either triplicate or quadruplicatecultures.

FIG. 7 shows the Ig production by either B cell line as a function ofthe concentration of DR7-primed T_(h) with optimal Ig production inducedat either 1:1 or 1:2 T_(h):B ratios. At T_(h):B ratios higher than 1:1inhibition of Ig production was observed. Hence, all further experimentswere carried out using a T_(h):B ratio of 1:2. As shown in FIG. 7, theseunprimed resting CD4 ⁺ T_(h) cells slightly induced 1gM production bySKW B cells but has no effect on the IgG production by CESS B cells in4-day cultures. This slight helper effect observed with unprimed CD4⁺CD45RO⁺ population during the Ig induction cultures. The production ofIg by CESS (IgG) or SKW (1gM) B cells induced by DR7-primed CD4 ⁺ T_(h)was specific for HLLA-DR7 because similarly activated DRw6-primed CD4 ⁺T_(h) (stimulated with DRw6 ⁺ ARENT B cells and autologous to theDR7-primed T_(h)) were unable to induce Ig production by either CESS orSKW B cells.

The roles of CD28 and B7 during cognate T_(h):B-induced Ig productionwere further examined using anti-CD28 and anti-B7 mAbs. Both CESS andSKW B cells constitutively express B7 antigen on their surface and thus,represent a source of unifonnly activated B cell populations for use inT_(h)-B cognate interactions or in cytokine-driven non-cognatematuration. Thus, DR7 ⁺ B cells (CESS or SKW) were cultured for 4 dayswith DR7-specific CD4 ⁺ T_(h) line at T_(h):B ratio of 1:2 and mAb toCD28 and B7, (and CD7 and CD57 as controls) were added to these culturesat different concentrations. Ig production (1gM, FIG. 8 a and IgG, FIG.8 b) at the end of 3-day cultures was quantitated in cell-free SN. FIG.8 shows that both anti-CD28 and anti-B7 mAbs but not theirisotype-matched mAb controls (anti-CD7 and anti-CD57, respectively)inhibited Tb induced Ig production by B cells in a does-dependentmanner. Once again, anti-CD28 mAb-mediated inhibition of Ig productionwas stronger than that by anti-B7 mAb. In contrast, Ig production byeither B cells induced by exogenous IL-6 (non-cognate differentiation)was not affected by any of the above mAb.

These results strongly suggest that the interaction between CD28 and B7,during cognate T_(h)-B collaboration, in addition to activation of T_(h)cells, is pivotal to the differentiation of activated B cells into Igsecreting cells.

The above results demonstrate the relationship of CD28 receptor and itsligand, the B7 antigen, as a co-stimulatory transmembranereceptor-ligand pair influencing T_(h):B interactions. Involvement ofboth CD28 and B7 during T_(h):B collaboration was demonstrated byinhibition by anti-CD28 and anti-B7 of not only T_(h) cell activationbut also T_(h)-induced differentiation of B cells into IgSC. It appearsas if the observed inhibitory effects of anti-CD28 and anti-B7 mAbs aredue to the inhibition of CD28:B7 interaction underlying these responses.

Interaction between CD28 receptor and B7 antigen may influence theproduction of cytokines and thus B cell differentiation. Ligation ofCD28 by B7 during T_(h):B collaboration may facilitate sustainedsynthesis and delivery of cytokines for their utilization during thedifferentiation of B cells into immunoglobulin secreting cells. The lackof inhibition by anti-CD28 and anti-B7 mAbs of cell dependentdifferentiation of CESS or SKW B cells induced with exogenous IL-4 orIL-6 suggests that CD28:B7 interaction controls either production ofthese cytokines, or their targeted delivery to B cells, or both of theseevents.

The interaction of CD28 and B7 is most likely not restricted to T_(h):Bcell interactions, and applies more generally to otherantigen-presenting cells such as monocyte/Mφ, dendritic cells, andepidermal Langerhans cells. Ligation of a nominal antigen presented inconjunction with MHC class II molecules on the surface ofantigen-presenting cells by the TcR/CD3 complex on the surface of T_(h)cells may lead to elevated expression of B7 antigen by these cells,which, via the interaction with CD28, then facilitates the production ofvarious cytokines by T_(h). This in turn drives both growth anddifferentiation of both T_(h) and B cells.

EXAMPLE 3

Characterization of the Interaction between CD28 Receptor and B7 Antigen

I. Preparation of Fusion Proteins

To further characterize the biochemical and functional aspects of theinteractions between the CD28 receptor and B7 antigen, fusion proteinsof B7 and CD28 with human immunoglobulin C gamma 1 (human Ig Cyl) chainswere constructed and expressed and used to measure the specificity andapparent affinity of interaction between these molecules. Purified B7Igfusion protein, and CHO cells transfected with B7 antigen were used toinvestigate the functional effects of this interaction on T cellactivation and cytokine production.

Preparation of B7Ig and CD28Ig Fusion Proteins

B7Ig and CD28Ig fusion proteins were prepared as follows. DNA encodingthe amino acid sequence corresponding to the extracellular domain of therespective protein (B7 and CD28) was joined to DNA encoding the aminoacid sequences corresponding to the hinge, CH2 and CH3 regions of humanimmunoglobulin Cγl. This was accomplished as follows.

Plasmid Construction. Expression plasmids were used containing cDNAencoding the amino acid sequence corresponding to CD28 (pCD28) asdescribed by Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84:8573 (1987),incorporated by reference, and provided by Drs. Aruffo and Seed, MassGeneral Hospital, Boston, Mass. Expression plasmids containing cDNAencoding the amino acid sequence corresponding to CD5 (pCD5) asdescribed by Aruffo, Cell 61:1303 (1990), and also provided by Dr.Aruffo, and eDNA encoding the amino acid sequence corresponding to B7(pB7) as described by Freeman et al., J. Immunol. 143:2714 (1989)) andprovided by Dr. Freeman, Dana Farber Cancer Institute, Boston, Mass.,were also used.

For initial attempts at expression of soluble forms of CD28 and B7,constructs were made (OMCD28 and OMB7) in which stop codons wereintroduced upstream of the transmembrane domains and the native signalpeptides were replaced with the signal peptide from oncostatin M (Maliket al., Mol. Cell Biol. 9:2847 (1989)). These were made using syntheticoligonucleotides for reconstruction (OMCD28) or as primers (OMB7) forPCR. OMCD28, is a CD28 cDNA modified for more efficient expression byreplacing the signal peptide with the analogous region from oncostatinM. CD28Ig and B7Ig fusion constructs were made in two parts. The 5′portions were made using OMCD28 and OMB7 as templates and theoligonucleotide, CTAGCCACTGAAGCTTCACCATGGGTGTACTGCTCACAC (SEQ ID NO:1)(corresponding to the oncostatin M signal peptide) as a forward primer,and either TGGCATGGOCTCCTGATCAGGCTTAGAAGGTCCGGGAAA (SEQ ID NO:2), or,TTTGGGCTCCTGATCAGGAAAATGCTCTTGCTTGGTTGT (SEQ ID NO:3) as reverseprimers, respectively. Products of the PGR reactions were cleaved withrestriction endonucleases (Hind III and Bcll) as sites introduced in thePCR primers and gel purified.

The 3′ portion of the fusion constructs corresponding to human Ig Cγlsequences was made by a coupled reverse transcriptase (from Avianmyeloblastosis virus; Life Sciences Associates, Bayport, N.Y.)-PCRreaction using RNA from a myeloma cell line producing human-mousechimeric mAb L6 (provided by Dr. P. Fell and M. Gayle, Bristol-MyersSquibb Pharmaceutical Research Institute, Seattle, Wash.) as template.The oligonucleotide, AAGCAAGAGCATTTTCCTGATCAGGAGCCCAAATCTTCTGACAAAACTCACACATCCCCACCGTCCCCAGCACCT GAACTCCTG (SEQ IDNO:4), was used as forward primer, andCTTCGACCAGTCTAGAAGCATCCTCGTGCGACGCGAGAGC (SEQ ID NO:5) as reverseprimer. Reaction products were cleaved with BclI and XbaI and gelpurified. Final constructs were assembled by ligating HindIII/BclIcleaved fragments containing CD28 or B7 sequences together withBcIL/XbaI cleaved fragment containing Ig Cγl sequences into HindIII/XbaIcleaved CDM8. Ligation products were transformed into MC1O6l/p3 E. colicells and colonies were screened for the appropriate plasmids. Sequencesof the resulting constructs were confirmed by DNA sequencing. The DNAused in the B7 construct encodes amino acids from about position 1 toabout position 215 of the sequence corresponding to the extracellulardomain of the B7 antigen, and for CD28, the DNA encoding amino acidsfrom about position 1 to about position 134 of the sequencecorresponding to the extracellular domain of the CD28 receptor.

CD5Ig was constructed in identical fashion, usingCATTGCACAGTCAAGCTTGCATGCCCATGGGTTCTCTGGCCACCTTG (SEQ ID NO:6), asforward primer and ATCCACAGTGCAGTGATCATTTGGATCCTGOCATGTGAC (SEQ ID NO:7)as reverse primer. The PCR product was restriction endonuclease digestedand ligated with the Ig Cγl fragment as described above. The resultingconstruct (CD5Ig) encodes an amino acid sequence containing residuesfrom about position 1 to about position 347 of CD5, two amino acidsintroduced by the construction procedure (amino acids DQ), followed bythe Ig Cγhinge region.

In initial attempts to make soluble derivatives of B7 and CD28, cDNAconstructs were made encoding molecules truncated at the NH₂-terminalside of their transmembrane domains. In both cases, the native signalpeptides were replaced with the signal peptide from oncostatin M (Malik,supra, 1989), which mediates efficient release of secreted proteins intransient expression assays. The cDNAs were cloned into an expressionvector, transfected into COS cells, and spent culture medium was testedfor secreted forms of B7 and CD28. In this fashion, several solubleforms of B7 were produced, but in repeated attempts, soluble CD28molecules were not detected.

The next step was to construct receptor Ig Cγl fusion proteins. The DNAsencoding amino acid sequences corresponding to B7 and CD28 extracellularregions, preceded by the signal peptide to oncostatin M, were fused inframe to an Ig Cγl cDNA, as shown in FIG. 9. During construction, the Ighinge disulfides were mutated to serine residues to abolish intrachaindisulfide bonding. The resulting fusion proteins were produced in COScells and purified by affinity chromatography on immobilized protein Aas described below. Yields of purified protein were typically 1.5-4.5mg/liter of spent culture medium.

Polymerase Chain Reaction (PCR). For PCR, DNA fragments were amplifiedusing primer pairs as described below for each fusion protein. PCRreactions (0.1 ml final volume) were run in Tap polymerase buffer(Stratagene, La Jolla, Calif.), containing 20 μmoles each of dNTP;50-100 μmoles of the indicated primers; template (1 ng plasmid or cDNAsynthesized from ≦1 μg total RNA using random hexamer primer, asdescribed by Kawasaki in PCR Protocols, Academic Press, pp. 21-27(1990), incorporated by reference herein); and Tap polymerase(Stratagene). Reactions were run on a thermocycler (Perkin Elmer Corp.,Norwalk, Conn.) for 16-30 cycles (a typical cycle consisted of steps of1 min at 94° C., 1-2 min at 50° C. and 1-3 min at 72° C.).

Cell Culture and Transfections. COS (monkey kidney cells) weretransfected with expression plasmids using a modification of theprotocol of Seed and Aruffo (Proc. Natl. Acad. Sci. 84:3365 (1987)),incorporated by reference herein. Cells were seeded at 10⁶ per 10 cmdiameter culture dish 18-24 h before transfection. Plasmid DNA was added(approximately 15 μg/dish) in a volume of 5 ml of serum-free DMEM™containing 0.1 mM cloroquine and 600 μg/ml DEAE Dextran™, and cells wereincubated for 3-3.5 h at 37° C. Transfected cells were then brieflytreated (approximately 2 min) with 10 % dimethyl sulfoxide in PBS andincubated at 37° C. for 16-24 h in DMEM™ containing 10 % FCS. At 24 hafter transfection, culture medium was removed and replaced withserum-free DMEM™ (6 ml/dish). Incubation was continued for 3 days at 37°C, at which time the spent medium was collected and fresh serum-freemedium was added. After an additional 3 days at 37° C., the spent mediumwas again collected and cells were discarded. CHO cells expressing CD28,CD5 or B7 were isolated as described by Linsley et al., (1991) supra. asfollows: Briefly, stable transfectants expressing CD28, CD5, or B7, wereisolated following cotransfection of dihydrofolate reductase-deficientChinese hamster ovary (dhfr⁻CHO) cells with a mixture of the appropriateexpression plasmid and the selectable marker, pSV2dhfr, as describedabove in Example 1. Transfectants were then grown in increasingconcentrations of methotrexate to a final level of 1 μM and weremaintained in DMEM™ supplemented with 10 % fetal bovine serum (FBS), 0.2mM proline and 1 μM methotrexate. CHO lines expressing high levels ofCD28 (CD28 ⁺ CHO) or B7 (B7 ⁺ CHO) were isolated by multiple rounds offluorescence-activated cell sorting (FACS^(R)) following indirectimmunostaining with mAbs 9.3 or BB-1. Amplified CHO cells negative forsurface expression of CD28 or B7 (dhfr⁺ CHO) were also isolated byFACS^(R) from CD28-transfected populations.

Immnunostaining and FACS^(R) Analysis. Transfected CHO cells oractivated T cells were analyzed by indirect immunostaining. Beforestaining, CHO cells were removed from their culture vessels byincubation in PBS containing 10 mM EDTA. Cells were first incubated withmurine mAbs 9.3 (hansen et al., Immunogenetics 10:247 (1980)) or BB-1(Yokochi et al., supra) at 10 μg/ml, or with Ig fusion proteins (CD28Ig,B7Ig, CD5Ig or chimeric mAb L6 containing Ig Cγl, all at 10 μg/ml inDMEM™ containing 10 % FCS) for 1-2 h at 4° C. Cells were then washed andincubated for an additional 0.5-2h at 4° C. with FITC-conjugated secondstep reagent (goat anti-mouse Ig serum for murine mAbs, or goatanti-human Ig Cγ serum for fusion proteins (Tago, Inc., Burlingame,Calif.). Fluorescence was analyzed on 10,000 stained cells using a FACSlV^(R) cell sorter (Becton Dickinson and Co., Mountain View, Calif.)equipped with a four decade logarithmic amplifier.

Purification of Ig Fusion Proteins. The first, second and thirdcollections of spent serum-free culture media from transfected COS cellswere used as sources for the purification of Ig fusion proteins. Afterremoval of cellular debris by low speed centrifugation, medium wasapplied to a column (approximately 200-400 ml medium/ml packed bedvolume) of immobilized protein A (Repligen Corp., Cambridge, Mass.)equilibrated with 0.05 M sodium citrate, pH 8.0. After application ofthe medium, the column was washed with 1 M potassium phosphate, pH 8,and bound protein was eluted with 0.05 M sodium citrate, pH 3. Fractionswere collected and immediately neutralized by addition of 1/10 volume of2 M Tris, pH 8. Fractions containing the peak of A₂₈₀ absorbing materialwere pooled and dialyzed against PBS before use. Extinction coefficientsof 2.4 and 2.8 ml/mg for CD28Ig and B7Ig, respectively, by amino acidanalysis of solutions of known absorbance. The recovery of purifiedCD28Ig and B7Ig binding activities were nearly quantitative as judged byFACS^(R) analysis after indirect fluorescent staining of B7 ⁺ and CD28 ⁺CHO cells.

SDS Page. SDS-PAGE was performed on liner acrylamide gradients gels withstacking gels of acrylamide. Aliquots (1 μg) B7Ig (lanes 1 and 3 of FIG.10) or CD28Ig (lanes 2 and 4) were subjected to SDS-PAGE (4-12 %acrylamide gradient) under nonreducing (-βME, lanes 1 and 2) or reducing(+βME, lanes 3 and 4) conditions. Lane 5 of FIG. 10 shows molecularweight (M_(r)) markers. Gels were stained with Coomassie Brilliant Blue,destained, and photographed or dried and exposed to X-ray film (Kodak™XAR−5; Eastman Kodak Co., Rochester, N.Y.) for autoradiography tovisualize proteins.

As shown in FIG. 10, the B7Ig fusion protein migrated during SDS-PAGEunder nonreducing conditions predominantly as a single species of M_(r)70,000, with a small amount of material migrating as a M, approximately150,000 species. After reduction, a single M_(r) approximately 75,000species was observed. CD28Ig migrated as a Mr approximately 140,000species under non-reducing conditions and a M_(r) approximately 70,000species after reduction, indicating that it was expressed as ahomodimer. Since the Ig Cγl hinge cysteines had been mutated, disulfidelinkage probably involved cysteine residues which naturally forminterchain bonds in the CD28 homodimer (Hansen et al., ImmunoRenetics10:247 (1980)).

DNAs encoding the amino acid sequences corresponding to the B7Ig fusionprotein and CD28Ig fusion protein have been deposited with the ATCC inRockville, Md., under the terms of the Budapest Treaty on May 31, 1991and there have been accorded accession Nos: 68627 (B7Ig) and 68628(CD28Ig).

II. Characterization of B7Ig and CD28Ig Cγl Fusion Proteins

To investigate the functional activities of B7Ig and CD28Ig, binding ofCHO cell lines expressing CD28 or B7 was tested as follows. In earlyexperiments, spent culture media from transfected COS cells was used asa source of fusion protein, while in later experiments, purifiedproteins were used (see FIG. 11).

Binding of B7Ig and CD28Ig to CHO cells. Binding of CD28Ig and B7Igfusion proteins was detected by addition of FITC-conjugated goatanti-human Ig second step reagent as described above. B7Ig was bound byCD28 ⁺ CHO, while CD28Ig was bound by B7 ⁺ CHO. B7Ig also bound weaklyto B7 ⁺ CHO (FIG. 11), suggesting that this molecule has a tendency toform homophilic interactions. No binding was detected of chimeric mAb L6containing human Ig Cγ1, or another fusion protein, CD5Ig. Thus B7Ig andCD28Ig retain binding activity for their respective counter-receptors.

The apparent affinity of interaction between B7 and CD28 was nextdetermined. B7Ig was either iodinated or metabolically labeled with[³⁵S] methionine, and radiolabeled derivatives were tested for bindingto immobilized CD28Ig or to CD28 ⁺ CHO cells.

Radiolabeling of B7Ig. Purified B7Ig (25 μg) in a volume of 0.25 ml of0.12 M sodium phosphate, pH 6.8 was jodinated using 2 mCi ¹²⁵I and 10 μgof chloramin T™. After 5 min at 23° C., the reaction was stopped by theaddition of 20 μg sodium metabisulfite, followed by 3 mg of KI and 1 mgof BSA. Iodinated protein was separated from untreated ¹²⁵I bychromatography on a 5ml column of Sephadex™ G-10 equilibrated with PBScontaining 10 % FCS. Peak fractions were collected and pooled. Thespecific activity of ¹²⁵I-B7Ig labeled in this fashion was 1.5×10⁶cpm/pmol.

B7Ig was also metabolically labeled with [35S]methionine. COS cells weretransfected with a plasmid encoding B7Ig as described above. At 24 hafter transfection, [³⁵S]methionine (<800 Ci/mmol; Amersham Corp.,Arlington Heights, Ill.) was added to concentrations of 115 μCi/ml) inDMEM™ containing 10 % FCS and 10 % normal levels of methionine. Afterincubation at 37° C. for 3 d, medium was collected and used forpurification of B7Ig as described above. Concentrations of[³⁵S]methionine-labeled B7Ig were estimated by comparison of stainingintensity after SDS-PAGE with intensities of known amounts of unlabeledB7Ig. The specific activity of [³⁵S]methionine-labeled B7Ig wasapproximately 2×10⁶ cpm/μg.

Binding Assays. For assays using immobilized CD28Ig, 96-well plasticdishes were coated for 16-24 h with a solution containing CD28Ig (0.5 μgin a volume of 0.05 ml of 10 mM Tris, pH 8). Wells were then blockedwith binding buffer (DMEM™ containing 50 mM BES, pH 6.8, 0.1 % BSA, and10 % FCS) (Sigma Chemical Co., St. Louis, Mo.) before addition of asolution (0.09 ml) containing ¹²⁵I-B7Ig (approximately 3×10⁶ cpm, 2×10⁶cpm/pmol) or [³⁵S]-B7Ig (1.5×10⁵ cpm) in the presence of absence ofcompetitor to a concentration of 24 nM in the presence of theconcentrations of unlabeled chimeric L6 mAb, mAb 9.3, mAb BB-1 or B7Ig,as indicated in FIG. 12. After incubation for 2-3 h at 23° C., wellswere washed once with binding buffer, and four times with PBS.

Plate-bound radioactivity was then solubilized by addition of 0.5 NNaOH, and quantified by liquid scintillation or gamma counting. In FIG.12, radioactivity is expressed as a percentage of radioactivity bound towells treated without competitor (7,800 cpm). Each point represents themean of duplicate determinations; replicates generally varied from themean by ≦20 %. Concentrations were calculated based on M_(r) of 75,000per binding site for mAbs and 51,000 per binding site for B7Ig. Whenbinding of ¹²⁵I-B7 to CD28 ⁺ CHO cells was measured, cells were seeded(2.5×10⁴/well) in 96-well plates 16-24 h before the start of theexperiment. Binding was otherwise measured as described above.

The results of a competition binding experiment using ¹²⁵I-B7Ig andimmobilized CD28Ig are shown in FIG. 12. Binding of ¹²⁵I-B7Ig wascompeted in dose-dependent fashion by unlabeled B7Ig, and by mAbs 9.3and BB-1. mAb 9.3 was the most effective competitor (half-maximalinhibition at 4.3 nM), followed by mAb BB-1 (half-maximal inhibition at140 nM) and B7 Ig (half-maximal inhibition at 280 nM). Thus, mAb 9.3 wasapproximately 65-fold more effective as a competitor than B7Ig,indicating that the mAb has greater apparent affinity for CD28. The samerelative difference in avidities was seen when [³⁵S]methionine-labeledB7Ig was used. Chimeric mAb L6 did not significantly inhibit binding.The inhibition at high concentrations in FIG. 12 was not seen in otherexperiments.

When the competition data shown in FIG. 12 were replotted in theScatchard representation (FIG. 13), a single class of binding sites wasobserved (binding constant (Kd) estimated from the slope of the linebest fitting the experimental data (r=−0.985), K_(d) of approximately200 nM. An identical K_(d) was detected for binding of ¹²⁵I-B7Ig to CD28⁺ CHO cells. Thus, both membrane bound CD28 and immobilized CD28Igshowed similar apparent affinities for ¹²⁵I-B7.

Binding of B7Ig to CD28 expressed on T cells

Although B7Ig bound to immobilized CD28Ig, and to CD28 ⁺ CHO cells, itwas not known whether B7Ig could bind to CD28 naturally expressed on Tcells. This is an important consideration since the level of CD28 ontransfected cells was approximately 10-fold higher than that found onPHA-activated T cells as shown above in Example 1. PHA-activated T cellswere prepared as follows.

Cell Separation and Stimulation. PBL were isolated by centrifugationthrough Lymphocyte Separation Medium™ (Litton Bionetics, Kensington,Md.) and cultured in 96-well, flat-bottomed plates (4×10⁴ cells/well, ina volume of 0.2 ml) in RPMI™ containing 10% FCS. Cellular proliferationof quadruplicate cultures was measured by uptake of [³H]thymidine duringthe last 5 h of a 3 day (d) culture. PHA-activated T cells were preparedby culturing PBL with 1 μg/ml PHA (Wellcome) for 5 d, and 1 d in mediumlacking PHA. Viable cells were collected by sedimentation throughLymphocyte Separation Medium™ before use.

PHA-activated T cells were then tested for binding of B7Ig (10 μg/ml) byFACS^(R) analysis after indirect immunofluorescence as described above.Where indicated (FIG. 14), mAbs 9.3 or BB-1 were also added at 10 μg/mlto cells simultaneously with B7Ig. Bound mAb was detected with aFITC-conjugated goat anti-human Ig Cγl reagent.

As shown in FIG. 14, these cells bound significant levels of B7Ig, andbinding was inhibited by mAbs 9.3 and BB-1.

The identity of B7Ig-binding proteins was also determined byimmunoprecipitation analysis of ¹²⁵I-surface labeled cells as follows.

Cell Surface Iodination and Immunoprecipitation. PHA-activated T cellswere cell-surface labeled with ¹²⁵I using lactoperoxidase and H₂O₂ asdescribed by Vitetta et al., J. Exp. Med. 134:242 (1971), incorporatedby reference herein. Aliquots of a nonionic detergent extract of labeledcells (approximately 3×10⁸ cpm in a volume of 0.12 ml) were prepared asdescribed by Linsley et al., J. Biol. Chem. 263: 8390 (1988),incorporated by reference herein, and subjected to immunoprecipitationanalysis and SDS-PAGE, as described above using a 5-15 % acrylamidegradient, under reducing (FIG. 15, +βMB, lanes 1-7) or non-reducingconditions (−βME, lanes 8 and 9), with no addition (lane 1), addition ofmAb 9.3 (5 μg, lane 2), addition of B7Ig (10 μg, lane 3), or addition ofchimeric L6 mAb (10 μg, lane 7).

As shown in FIG. 15, Both mAb 9.3 and B7Ig immunoprecipitated a proteinhaving a M_(r) of approximately 45,000 under reducing conditions, andproteins having a M_(r) of approximately 45,000 and approximately 90,000under nonreducing conditions, with the latter form being more prominent.The protein having a M_(r) of approximately 45,000 found in the sampleprecipitated with chimeric mAb L6 was due to spillover and was notobserved in other experiments. mAb 9.3 was more effective atimmunoprecipitation than B7Ig, in agreement with the greater affinity ofthe mAb (FIGS. 12 and 13). Identical results were obtained when CD28 ⁺CHO cells were used for immunoprecipitation analysis. Preclearing ofCD28 by immunoprecipitation with mAb 9.3 also removed B7Ig-precipitablematerial, indicating that both mAb 9.3 and B7Ig bound the same¹²⁵I-labeled protein.

Taken together, the results in these experiments indicate that CD28 isthe major receptor for B7Ig on PHA-activated T cells.

Effects of B7 Binding to CD28 on CD28-mediated Adhesion

mAbs to CD28 have potent biological activities on T cells, suggestingthat interaction of CD28 with its natural ligand(s) may also haveimportant functional consequences. As a first step in determiningfunctional consequences of interaction between B7 and CD28, it wasdetermined whether B7Ig could block the CD28-mediated adhesion assaydescribed above. The adhesion of ⁵¹Cr-labeled PM lymphoblastoid cells tomonolayers of CD28 ⁺ CHO cells was measured as described above, in thepresence of the indicated amounts of mAb 9.3 or B7Ig. Data are expressedin FIG. 16 as a percentage of cells bound in the absence of competitor(40,000 cpm or approximately 1.1×10⁵ cells). Each point represents themean of triplicate determinations; coefficients of variation were ≦25 %.

As shown in FIG. 16, B7Ig blocked CD28-mediated adhesion somewhat lesseffectively than mAb 9.3 (half-maximal inhibition at 200 nM as comparedwith 10 nM for mAb 9.3). The relative effectiveness of these moleculesat inhibiting CD28-mediated adhesion was similar to their relativebinding affinities in competition binding experiments (FIG. 12). CD28Igfailed to inhibit CD28-mediated adhesion at concentrations of up to 950nM, suggesting that much higher levels of CD28Ig were required tocompete with the high local concentrations of CD28 present ontransfected cells.

The Effects of B7 On T Cell Proliferation

It was further investigated whether triggering of CD28 by B7 wascostimulatory for T cell proliferation. The ability of B7Ig tocostimulate proliferation of PBL together with anti-CD3 was firstexplored. PBL were isolated and cultured in the presence of thecostimulators of T cell proliferation indicated in Table 2. Anti-CD3stimulation was with mAb G19-4 at 1 μg/ml in solution. For CD28stimulation, mAb 9.3 or B7Ig were added in solution at 1 μg/ml, or afterimmobilization on the culture wells by pre-incubation of proteins at 10μg/ml in PBS for 3 h at 23° C. and then washing the culture wells. B7⁺CHO and control dhfr⁺CHO cells were irradiated with 1,000 rad beforemixing with PBL at a 4:1 ratio of PBL/CHO cells. After culture for 3 d,proliferation was measured by uptake of [^(H)] thymidine for 5 h. Valuesshown are means of determinations from quadruplicate cultures (SEM<15%).

In several experiments, B7Ig in solution at concentrations of 1-10 μg/mlshowed only a modest enhancement of proliferation even though theanti-CD28 mAb 9.3 was effective. Because CD28 crosslinking has beenidentified as an important determinant of CD28 signal transduction(Ledbetter et al., Blood 75:1531 (1990)), B7Ig was also compared to 9.3when immobilized on plastic wells (Table 2, Exp. 1).

TABLE 2 [³H]-T incorporation −Anti-CD3 +Anti-CD3 Exp. 1 CD28 Stimulationcpm × 10⁻³ 1 None 0.1 26.0 mAb 9.3 (soln.) 0.3 156.1 mAb 9.3 (immob.)0.1 137.4 B7Ig (immob.) 0.1 174.5 2 None 0.2 19.3 mAb 9.3 (soln.) 0.475.8 B7 + CHO cells 9.4 113.9 dhfr + CHO cells 23.8 22.1

Under these conditions, B7Ig was able to enhance proliferation andcompared favorably with mAb 9.3. B7 ⁺ CHO cells also were tested andcompared with control dhfr⁺ CHO cells for costimulatory activity onresting lymphocytes (Table 2, Exp. 2). In this experiment, proliferationwas seen with dhfr⁺ CHO cells in the absence of anti-CD3 mAb because ofresidual incorporation of [³H]thymidine after irradiation of thesecells. The stimulation by dhfr⁺ cells was not enhanced by anti-CD3 mAband was not observed in other experiments (Tables 3 and 4) wheretransfected CHO cells were added at lower ratios.

For the experiments shown in Table 3, PHA blasts were cultured at 50,000cells/well with varying amounts of irradiated CH) cell transfectants.After 2 d of culture, proliferation was measured by a 5 h pulse of[³H]thymidine. Shown are means of quadruplicate determinations (SEM<15%). Background proliferation of PHA blasts without added CHO cells was11,200 cpm. [³H]thymidine incorporation by irradiated B7 ⁺ CHO and CD5 ⁺CHO cells alone was >1,800 cpm at each cell concentration and wassubtracted from the values shown. For the experiments summarized inTable 4, PHA blasts were stimulated as described in Table 3, withirradiated CHO cells at a ratio of 40:1 T cells/CHO cells. mAbs wereadded at 10 μg/ml at the beginning of culture. mAb LB-1 (Yokochi et al.,supra) is an isotype-matched control for mAb BB-1. Proliferation wasmeasured by uptake of [³H]thymidine during a 5 h pulse after 2 d ofculture. Values represent means of quadruplicate cultures (SEM<15 %).

B7 ⁺ CHO cells were very effective at costimulation with anti-CD3 mAb,indicating that cell surface B7 had similar activity in this assay asthe anti-CD28 mAbs.

B7 ⁺ CHO cells were also tested as to whether they could directlystimulate proliferation of resting PHA blasts which respond directly toCD28 crosslinking by mAb 9.3. Again, the B7 ⁺ CHO cells were very potentin stimulating proliferation (Table 3) and were able to do so at verylow cell numbers (PHA blast:B7 ⁺ CHO ratios of >800:1). The control CD5⁺ CHO cells did not possess a similar activity. (In a number ofdifferent experiments neither dhfr CHO, CD5 ⁺ CHO, nor CD7 ⁺ CHO cellsstimulated T cell proliferation.

These were therefore used interchangeably as negative controls foreffects induced by B7 ⁺ CHO cells. The stimulatory activity of B7 ⁺ CHOwas further shown to result from CD28/B7 interaction, since mAb BB1inhibited stimulation by the B7 ⁺ CHO cells without affecting backgroundproliferation in the presence of CD7 ⁺ CHO cells (Table 4). mAb LB-1(Yokochi et al., supra), an 1gM mAb to a different B cell antigen, didnot inhibit proliferation. mAb 9.3 (Fab fragments) inhibitedproliferation induced by B7 ⁺ CHO and as well as backgroundproliferation seen with CD7 ⁺CHO cells.

TABLE 3 [³H]-T incorporation +B7⁺ CHO +CD5⁺ CHO T cells/CHO cells cpm ×10⁻³  25:1 92.7 15.5  50:1 135.4 19.4 100:1 104.8 16.8 200:1 90.3 17.7400:1 57.0 13.7 800:1 42.3 17.6

TABLE 4 mAb [³H]-T incorporation Stimulation cpm × 10⁻³ None None 10.8B7⁺CHO None 180 B7⁺CHO 9.3 Fab 132 B7⁺CHO BB-1 98.3 B7⁺CHO LB-1 196 CD7⁺CHO None 11.5 CD7⁺ CHO 9.3 Fab 10.0 CD7⁺ CHO BB-1 10.0 CD7⁺ CHO LB-111.3

These experiments show that B7 is able to stimulate signal transductionand augment T cell activity by binding to CD28, but that crosslinking isrequired and B7 expressed on the cell surface is most effective.

The Effects of B7 on IL-2 mRNA Accumulation

Effects of CD28/B7 interactions on IL-2 production were investigated byanalyzing transcript levels in PHA-blasts stimulated with B7 ⁺ CHO cellsor CD7 ⁺ CHO cells. RNA was prepared from stimulated cells and tested byRNA blot analysis for the presence of IL-2 transcripts as follows.

PHA blasts (5×10⁷) were mixed with transfected CHO cells at a ratio of40:1 T cells/CHO cells, and/or mAbs as indicated in FIG. 17. mAb 9.3 wasused at 10 μg/ml. mAb BB-1 was added at 20 μg/ml 1 h before addition ofB7 ⁺ CHO cells. When mAb 9.3 was crosslinked, goat anti-mouse Ig (40μg/ml) was added 10 min after addition of mAb 9.3. Cells were incubatedfor 6 h at 37° C. and RNA was isolated and subjected to RNA blotanalysis using ³²p-labeled IL-2 or GAPDH probes as described below.

RNA was prepared from stimulated PHA blasts by the procedure describedby Chomczynki and Sacchi, Anal. Biochem. 162:156 (1987), incorporated byreference herein. Aliquots of RNA (20 μg) were fractionated onformaldehyde agarose gels and then transferred to nitrocellulose bycapillary action. RNA was crosslinked to the membrane by UVlight in aStratalinker™ (Stratagene, San Diego, Calif.), and the blot wasprehybridized and hybridized with a ³²P-labeled probe for human IL-2(prepared from an approximately 600-bp cDNA fragment provided by Dr. S.Gillis; lmmunex Corp., Seattle, Wash.). Equal loading of RNA samples wasverified both by rRNA staining and by hybridization with a ratglyceraldehyde-6-phosphate dehydrogenase probe (GAPDH, an approximately1.2-kb cDNA fragment provided by Dr. A. Purchio, Bristol-Myers SquibbPharmaceutical Research Institute, Seattle, Wash.).

As shown in FIG. 17, B7 ⁺ CHO cells, but not CD7 ⁺ CHO cells, inducedaccumulation of IL-2 mRNA transcripts. Induction by B7 ⁺ CHO cells waspartially blocked by mAb BB-1. Induction by B7 ⁺ CHO cells was slightlybetter than achieved by mAb 9.3 in solution, but less effective than mAb9.3 after crosslinking with goat anti-mouse Ig. Thus, triggering of CD28by cell surface B7 on apposing cells stimulated IL-2 mRNA accumulation.

The apparent K_(d) value for the interaction of soluble Ig Cγfusions ofCD28 and B7 (approximately 200 nM), obtained from the above experiments,is within the range of affinities observed for mAbs (2-10,000 nM; Alzariet al., Annu. Ref. J. Immunol. 6:555 (1988)) and compares favorably withthe affinities estimated for other lymphoid adhesion molecules. Schnecket al., (Cell 56:47 (1989)) estimated the affinity (K_(d) approximately100 nM) between a murine T cell hybridoma TCR and soluble alloantigen(class I MHC molecules). A K_(d) of 400 nM was measured between CD2 andLFA3 (Recny et al., J. Biol. Chem. 265:8542 (1990)). The affinity of CD4for class II MHC, while not measured directly, was estimated (Clayton etal., Nature (Lond.) 339:548 (1989)) to be≧10,000 times lower than theaffinity of gp120-CD4 interactions (K_(d)=4 nM; Lasky et al., Cell50:975 (1987)). Thus, the affinity of B7 for CD28 appears greater thanaffinities reported for some other lymphoid adhesion systems.

The degree to which the apparent K_(d) of CD28/B7 interaction reflectstheir true affinity, as opposed to their avidity, depends on the valencyand/or aggregation of the fusion protein preparations. The degree ofaggregation of these preparations was examined by size fractionation(TSK G3000SW column eluted with PBS). Under these conditions, B7Igeluted at M_(r) approximately 350,000, and CD28Ig at M_(r) approximately300,000. Both proteins thus behaved in solution as larger molecules thanthey appeared by SDS-PAGE (FIG. 10), suggesting that they may formhigher aggregates. Alternatively, these results may indicate that bothfusion proteins assume extended conformations in solution, resulting inlarge Stokes radii. Regardless, the interaction that was measured usingsoluble proteins probably underestimates the true avidity between CD28and B7 in their native membrane-associated state.

The relative contribution of different adhesion systems to the overallstrength of T cell-B cell interactions is not easily gauged, but islikely a function of both affinity/avidity and the densities on apposingcell surfaces of the different receptors and counter-receptors involved.Since both CD28 and B7 are found at relatively low levels on restinglymphoid cells (Lesslauer et al., Eur. J. Immuno. 16:1289 (1986);Freeman et al., supra 1989), they may be less involved than otheradhesion systems (Springer Nature (Land). 346:425 (1990)) in initiatingintercellular interactions. The primary role of CD28/B7 interactions maybe to maintain or amplifier a response subsequent to induction of thesecounter-receptors on their respective cell types.

Binding of B7 to CD28 on T cells was costimulatory for T cellproliferation (Tables 2-4) suggesting that some of the biologicaleffects of anti-CD28 mAbs result from their ability to mimic T cellactivation resulting from natural interaction between CD28 and itscounter-receptor, B7. mAb 9.3 has greater affinity for CD28 than doesB7Ig (FIGS. 15 and 16), which may account for the extremely potentbiological effects of this mAb (June et al., supra 1989) incostimulating polyclonal T cell responses. Surprisingly, however,anti-CD28 mAbs are inhibitory for antigen-specific T cell responses(Damle et al., Proc. Nati. Acad. Sci. USA 78:5096 (1981); Lesslauer etal., supra 1986). This may indicate that antigen-specific T cellresponses are dependent upon costimulation via CD28/B7 interactions, andthat inhibition therefore results from blocking of CD28 stimulation.Despite the inhibition, CD28 must be bound by mAb under theseconditions, implying that triggering by mAb is not always equivalent totriggering by B7. Although mAb 9.3 has higher apparent affinity for CD28than B7 (FIG. 12), it may be unable under these circumstances to inducethe optimal degree of CD28 clustering (Ledbetter et al., supra 1990) forsimulation.

CD28/B7 interactions may also be important for B cell activation and/ordifferentiation. As described above in Example 2, mAbs 9.3 and BB-1block Tb cell-induced Ig production by B cells. This blocking effect maybe due in part to inhibition by these mAbs of production of Tb-derived Bcell-directed cytokines, but may also involve inhibition of B cellactivation by interfering with direct signal transduction via B7. Theseresults suggest that cognate activation of B lymphocytes, as well asT_(h) lymphocytes, is dependent upon interaction between CD28 and B7.

The above results demonstrate that the ligand for CD28 receptor, the B7antigen, is expressed on activated B cells and cells of other lineages.These results also show that CD28 receptor and its ligand, B7, play apivotal role during both the activation of CD4 ⁺ T_(h) cell andT_(h)-induced differentiation of B cells. The inhibition of anti-CD28and anti-B7 mAbs on the cognate T_(h):B interaction also provide thebasis for employing the CD28Ig and B7Ig fusion proteins, and monoclonalantibodies reactive with these proteins, to treat various autoimmuneorders associated with exaggerated B cell activation such asinsulin-dependent diabetes mellitus, myasthenia gravis, rheumatoidarthritis and systemic lupus erythematosus (SLE).

As will be apparent to those skilled in the art to which the inventionpertains, the present invention may be embodied in forms other thanthose specifically disclosed above without departing from the spirit oressential characteristics of the invention. The particular embodimentsof the invention described above, are, therefore, to be considered asillustrative and not restrictive. The scope of the present invention isas set forth in the appended claims rather than being limited to theexamples contained in the foregoing description.

1. A method for blocking binding of B7 positive B cells to CD28 antigento inhibit CD28 specific T cell interaction comprising contacting B7positive B cells with an antibody or fragment thereof which binds B7antigen, wherein the B7 antigen consists of amino acid residues fromposition 1 to position 216 of SEQ ID NO.
 8. 2. A method for blocking thebinding of B7 antigen on B7-positive B cells to CD28 comprisingcontacting B7 positive cells with an antibody or fragment thereof whichbinds with said B7 antigen, wherein said antibody or fragment thereofbinds SEQ ID No.
 8. 3. A method for inhibiting an immune responsecomprising blocking the binding of B7 antigen on B7-positive B cells toCD28 by reacting B7 positive cells with an antibody or fragment thereofwhich binds with B7 antigen, wherein said B7 antigen consists of aminoacid residues from position 1 to position 216 of SEQ ID NO.
 8. 4. Amethod for inhibiting an immune response comprising blocking the bindingof B7 antigen on B7-positive B cells to CD28 by reacting B7 positivecells with an antibody of fragment thereof which binds with said B7antigen, wherein said antibody, or fragment thereof, binds SEQ ID NO. 8.5. The method of claim 1, 2, 3, 4, wherein said antibody is a monoclonalantibody.
 6. The method of claim 1, 2, 3, 4, wherein the fragment of theantibody is a Fab or F(ab′)2 fragment.
 7. A method for blocking bindingof B7 positive B cells to CD28 antigen to inhibit CD28 specific T cellinteraction comprising contacting B7 antigen positive B cells with amonoclonal antibody, or fragment thereof, which binds with B7 positive Bcells in sufficient amounts effective to prevent binding of said CD28antigen to said B7 antigen, wherein said B7 antigen consists of aminoacid residues from position 1 to position 216 of SEQ ID NO. 8.